26934, a novel cytidine deaminase-like molecule and uses thereof

ABSTRACT

Novel cytidine deaminase-like polypeptides, proteins, and nucleic acid molecules are disclosed. In addition to isolated, full-length cytidine deaminase-like proteins, the invention further provides isolated cytidine deaminase-like fusion proteins, antigenic peptides, and anti-cytidine deaminase-like antibodies. The invention also provides cytidine deaminase-like nucleic acid molecules, recombinant expression vectors containing a nucleic acid molecule of the invention, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which an cytidine deaminase-like gene has been introduced or disrupted. Diagnostic, screening, and therapeutic methods utilizing compositions of the invention are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/188,294 filed on Mar. 10, 2000, the contents of whichare hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] The invention relates to novel cytidine deaminase-like nucleicacid sequences and proteins. Also provided are vectors, host cells, andrecombinant methods for making and using the novel molecules.

BACKGROUND OF THE INVENTION

[0003] Cytidine deaminases convert, by irreversible hydrolyticdeamination, cytidine and deoxycytidine to uridine and deoxyuridine,respectively. The deamination of cytidine has been demonstrated toaffect numerous mammalian cellular processes. For instance, increasingevidence suggests that base-modification of mRNA editing occurs throughdeamination via cytidine deaminases. Current data suggests thatunregulated editing or inappropriate occurrence of mRNA editing can leadto various disease states. For a review see, for example, Smith et al.(1996) Trends in Genetics 12:418-424. Among vertebrates, examples ofmRNA editing resulting in a cytidine to uridine conversion include APOBmRNA in rat liver and intestine (Chen et al. (1987) Science 328:363-366and Powell et al. (1987) Cell 50:831-840); Neurofibromatosis type-1 mRNAin human tumors (Skuse et al. (1996) Nucleic Acid Res. 24:478-486);tRNA^(ASP) in Rat liver (Beier et al. (1992) Nucleic Acid Research20:2679-2683); and tRNA^(GLY) in marsupials (Janke et al. (1993) NucleicAcid 21:1523-1525).

[0004] The mRNA editing process of the APOB mRNA has been wellcharacterized. The base modification of APOB mRNA occurs throughhydrolytic removal of the C4 and C6 amino groups from cytidine. Theediting event results in two isoforms of the protein, APOB100 andAPOB48. The APOB protein binds lipid to form lipoprotein particles and,thereby, participates in the transports of lipids to peripheral bodytissues. Lipoprotein particles assembled on APOB48 are cleared from theblood more rapidly than those containing APOB100 and are not metabolizedto the low density lipoprotein (LDL). The cytidine deamination event ofthe APOB mRNA therefore influences the level of LDL in the blood streamand may ultimately influence the development of atherogenic diseases(Davison et al. (1993) Ann. Med. 25:539-543 and Greeve et al. (1993) J.Lipid Res. 34:1367-1383).

[0005] Biochemical analysis of the APOB mRNA editing process hasdemonstrate that the cytidine deaminase, APOBEC1 (APOB-editing catalyticsubunit 1), plays a critical role in the deamination event. Currentevidence suggests that APOBEC1 alone can only deaminate cytidinemononucleotides (Navaratnam et al. (1993) J. Biol. Chem.268:20709-20712). It appears that the ability to edit APOB mRNA isimposed upon APOCEC1 allosterically through its assembly with otherprotein (auxiliary factors) as holoenzymes editing complexes or“editosomes”.

[0006] Other cytidine deamination mRNA editing events have beenimplicated in cell cycle regulation. The editing site of NF1 mRNA is 67%identical to that found in APOB mRNA. A cytidine to uridine conversionin the NF1 mRNA results in a truncated translation product. Thetruncated product is unable to maintain RAS in the inactive GDP form,and would therefore, presumably, be unable to regulate cellproliferation (Skuse et al. (1996) Nucleic Acid Research 24:478-486).

[0007] In addition to its natural substrates, cytidine anddeoxycytidine, cytidine deaminases also catalyzes the deamination ofcytosine nucleoside analogs including the antineoplastic agents ARA-C,dFdC, and 5-AZA-CdR. The deamination of these compounds results in aloss of their pharmacological activity. Cytidine deaminases maytherefore have the ability to confer resistance to these drugs andpotentially circumvent the hematopoietic toxicity produced bychemotherapy with cytosine nucleoside analogs.

[0008] Recently, in vitro data has demonstrated that it is possible toconfer drug resistance to cytosine nucleoside analogs by the transfer ofthe cytidine deaminase gene into murine hematopoietic cells. Further invivo studies have demonstrated that is possible to transplant cytidinedeaminase-transduced marrow cells into mice and obtain long-termincreased expression of cytidine deaminase in hematopoietic cells. For areview see Bertion et al. (1999) Prog Exp Tum Res 36:124-142. Therefore,cytidine deaminases have potential to render normal hematopoietic cellsresistant to cytosine nucleoside analogs and thereby increase theclinical efficacy of these chemotherapeutic drugs in cancer treatment.

[0009] Cytidine deaminase family members play critical regulatory rolesin various cellular processes. Accordingly, it is valuable to the fieldof pharmaceutical development to identify and characterize novelcytidine deaminases. The present invention advances the state of the artby providing a novel human cytidine deaminase-like nucleic acid andpolypeptide.

SUMMARY OF THE INVENTION

[0010] Isolated nucleic acid molecules corresponding to cytidinedeaminase-like nucleic acid sequences are provided. Additionally, aminoacid sequences corresponding to the polynucleotides are encompassed. Inparticular, the present invention provides for isolated nucleic acidmolecules comprising nucleotide sequences encoding the amino acidsequences shown in SEQ ID NO:2. Further provided are cytidinedeaminase-like polypeptides having an amino acid sequence encoded by anucleic acid molecule described herein.

[0011] The present invention also provides vectors and host cells forrecombinant expression of the nucleic acid molecules described herein,as well as methods of making such vectors and host cells and for usingthem for production of the polypeptides or peptides of the invention byrecombinant techniques.

[0012] The cytidine deaminase-like molecules of the present inventionare useful as modulating agents in a variety of cellular processesincluding cytidine deamination and mRNA editing. Accordingly, in oneaspect, this invention provides isolated nucleic acid molecules encodingcytidine deaminase-like proteins or biologically active portionsthereof, as well as nucleic acid fragments suitable as primers orhybridization probes for the detection of cytidinedeaminase-like-encoding nucleic acids.

[0013] Another aspect of this invention features isolated or recombinantcytidine deaminase-like proteins and polypeptides. Preferred cytidinedeaminase-like proteins and polypeptides possess at least one biologicalactivity possessed by naturally occurring cytidine deaminase-likeproteins.

[0014] Variant nucleic acid molecules and polypeptides substantiallyhomologous to the nucleotide and amino acid sequences set forth in thesequence listings are encompassed by the present invention.Additionally, fragments and substantially homologous fragments of thenucleotide and amino acid sequences are provided.

[0015] Antibodies and antibody fragments that selectively bind thecytidine deaminase-like polypeptides and fragments are provided. Suchantibodies are useful in detecting the cytidine deaminase-likepolypeptides as well as in regulating cytidine deamination events, suchas those that occur during mRNA editing.

[0016] In another aspect, the present invention provides a method fordetecting the presence of cytidine deaminase-like activity or expressionin a biological sample by contacting the biological sample with an agentcapable of detecting an indicator of cytidine deaminase-like activitysuch that the presence of cytidine deaminase-like activity is detectedin the biological sample.

[0017] In yet another aspect, the invention provides a method formodulating cytidine deaminase-like activity comprising contacting a cellwith an agent that modulates (inhibits or stimulates) cytidinedeaminase-like activity or expression such that cytidine deaminase-likeactivity or expression in the cell is modulated. In one embodiment, theagent is an antibody that specifically binds to cytidine deaminase-likeprotein. In another embodiment, the agent modulates expression ofcytidine deaminase-like protein by modulating transcription of ancytidine deaminase-like gene, splicing of an cytidine deaminase-likemRNA, or translation of an cytidine deaminase-like mRNA. In yet anotherembodiment, the agent is a nucleic acid molecule having a nucleotidesequence that is antisense to the coding strand of the cytidinedeaminase-like mRNA or the cytidine deaminase-like gene.

[0018] In one embodiment, the methods of the present invention are usedto treat a subject having a disorder characterized by aberrant cytidinedeaminase-like protein activity or nucleic acid expression byadministering an agent that is an cytidine deaminase-like modulator tothe subject. In one embodiment, the cytidine deaminase-like modulator isan cytidine deaminase-like protein. In another embodiment, the cytidinedeaminase-like modulator is an cytidine deaminase-like nucleic acidmolecule. In other embodiments, the cytidine deaminase-like modulator isa peptide, peptidomimetic, or other small molecule.

[0019] The present invention also provides a diagnostic assay foridentifying the presence or absence of a genetic lesion or mutationcharacterized by at least one of the following: (1) aberrantmodification or mutation of a gene encoding an cytidine deaminase-likeprotein; (2) misregulation of a gene encoding an cytidine deaminase-likeprotein; and (3) aberrant post-translational modification of an cytidinedeaminase-like protein, wherein a wild-type form of the gene encodes aprotein with an cytidine deaminase-like activity.

[0020] In another aspect, the invention provides a method foridentifying a compound that binds to or modulates the activity of ancytidine deaminase-like protein. In general, such methods entailmeasuring a biological activity of an cytidine deaminase-like protein inthe presence and absence of a test compound and identifying thosecompounds that alter the activity of the cytidine deaminase-likeprotein.

[0021] The invention also features methods for identifying a compoundthat modulates the expression of cytidine deaminase-like genes bymeasuring the expression of the cytidine deaminase-like sequences in thepresence and absence of the compound.

[0022] Other features and advantages of the invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIGS. 1A and 1B provide the nucleotide (SEQ ID NO:1 or 3) andamino acid (SEQ ID NO:2) sequence for clone 26934.

[0024]FIG. 2 depicts a hydropathy plot of a human cytidinedeaminase-like molecule. Relative hydrophobic residues are shown abovethe dashed horizontal line, and relative hydrophilic residues are belowthe dashed horizontal line. The cysteine residues (cys) and Nglycosylation site (Ngly) are indicated by short vertical lines justbelow the hydropathy trace. The numbers corresponding to the amino acidsequence (shown in SEQ ID NO:2) of human cytidine deaminase-likemolecule are indicated. Polypeptides of the invention include fragmentswhich include: all or a part of a hydrophobic sequence (a sequence abovethe dashed line); or all or part of a hydrophilic fragment (a sequencebelow the dashed line). Other fragments include a cysteine residue or asN-glycosylation site.

[0025]FIG. 3 depicts an alignment of the cytidine and deoxycytidylatedeaminase zinc-binding region domain of human cytidine deaminase-likemolecule with a consensus amino acid sequence derived from a hiddenMarkov model. The upper sequence is the consensus amino acid sequence(SEQ ID NO:4), while the lower amino acid sequence corresponds to aminoacids 80 to 149 of SEQ ID NO:2.

[0026]FIG. 4 shows the expression of the 26934 mRNA in various tissuesand cell lines. The level of 26934 mRNA was analyzed in the followingtissues from left to right: artery normal; aorta diseased; vein normal;coronary SMC (smooth muscle cells); HUVEC (human umbilical veinendothelial cells); hemangioma; heart normal; heart CHF; kidney;skeletal muscle; adipose normal; pancreas; primary osteoblasts;osteoclasts (differentiated); skin normal; spinal cord normal; braincortex normal; brain hypothalamus normal; nerve; DRG (dorsal rootganglion); breasts normal; breast tumor; ovary normal; ovary tumor;prostate normal; prostate tumor; salivary glands; colon normal; colontumor; lung normal; lung tumor; lung COPD (chronic obstructive pulmonarydisease); colon IBD (inflammatory bowel disease); liver normal; liverfibrosis; spleen normal; tonsil; tonsil normal; lymph node normal; smallintestine normal; macrophages; synovium; BM-MNC (bone marrow—mononuclearcells); activated PBMC (peripheral blood mononuclear cells) (resting);neutrophils; megakaryocytes; and erythroid.

[0027]FIG. 5 shows the expression of the 26934 mRNA in various tissuesand cell lines. The level of the 26934 mRNA was analyzed in thefollowing tissues from left to right: normal breast (columns 1-3);tumorous breast tissue (columns 4-9); lymph node (breast met) (column10); lung (breast met) (column 11); ovary normal (columns 12-13);tumorous ovary (columns 14-18); normal lung (columns 19-21); tumorouslung (columns 22-27); normal colon (columns 28-30); tumorous colon(columns 31-34); colon-liver metastasis (columns 35-36); normal liver(column 37); cervix squamous CC (columns 38-39); HMVEC (columns 40-41);normal prostate (columns 42-43); and tumorous prostate (columns 44-45).

[0028]FIG. 6 shows the expression levels of the 26934 mRNA in normalovarian epithelial (NOE) cells and in clinical ovarian ascites samples.

[0029]FIG. 7 shows the level of 26934 mRNA expression in HEY cells, 0hours, 1 hour, 3 hours, 6 hours, 9 hours, and 12 hours after serumaddition to the HEY cells.

[0030]FIG. 8 shows the expression level of the 26934 mRNA in the ovariancells SKOV-3 following the addition of growth factor EGF (epidermalgrowth factor) and Hrg (Heregulin).

[0031]FIG. 9 shows the expression level of the 26934 mRNA in the ovariancells SKOV-3/variant cells following the addition of growth factor EGF(epidermal growth factor) and Hrg (Heregulin).

[0032]FIG. 10 summarizes the expression levels of the 26934 mRNA invarious ovarian cell lines.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention provides cytidine deaminase-like molecules.By “cytidine deaminase-like molecules” is intended a novel humansequence referred to as 26934, and variants and fragments thereof. Thesefull-length gene sequences or fragments thereof are referred to as“cytidine deaminase-like” sequences, indicating they share sequencesimilarity with cytidine deaminase genes. Isolated nucleic acidmolecules comprising nucleotide sequences encoding the 26934 polypeptidewhose amino acid sequence is given in SEQ ID NO:2, or a variant orfragment thereof, are provided. A nucleotide sequence encoding the 26934polypeptide is set forth in SEQ ID NO: 1 or 3. The sequences are membersof the cytidine deaminase family.

[0034] A novel human cytidine deaminase-like gene sequence, referred toas 26934. This gene sequence and variants and fragments thereof areencompassed by the term “cytidine deaminase-like” molecules or sequencesas used herein. The cytidine deaminase-like sequences find use inmodulating a cytidine deaminase-like function. By “modulating” isintended the upregulating or downregulating of a response. That is, thecompositions of the invention affect the targeted activity in either apositive or negative fashion.

[0035] The disclosed invention relates to methods and compositions forthe modulation, diagnosis, and treatment of a variety of disorders.Disorders of interest include, for example, cellular proliferativeand/or differentiative disorders including cancer, e.g., carcinoma,sarcoma, metastatic disorders or hematopoietic neoplastic disorders,e.g., leukemias. A metastatic tumor can arise from a multitude ofprimary tumor types, including but not limited to those of ovary,cervix, prostate, colon, lung, breast and liver origin.

[0036] As used herein, the terms “cancer”, “hyperproliferative” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state or condition characterized by rapidlyproliferating cell growth. Hyperproliferative and neoplastic diseasestates may be categorized as pathologic, i.e., characterizing orconstituting a disease state, or may be categorized as non-pathologic,i.e., a deviation from normal but not associated with a disease state.The term is meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. “Pathologic hyperproliferative” cells occur in diseasestates characterized by malignant tumor growth. Examples ofnon-pathologic hyperproliferative cells include proliferation of cellsassociated with wound repair.

[0037] The terms “cancer” or “neoplasms” include malignancies of thevarious organ systems, such as affecting lung, breast, thyroid,lymphoid, gastrointestinal, and genito-urinary tract, as well asadenocarcinomas which include malignancies such as most colon cancers,renal-cell carcinoma, prostate cancer and/or testicular tumors,non-small cell carcinoma of the lung, cancer of the small intestine andcancer of the esophagus.

[0038] The term “carcinoma” is art recognized and refers to malignanciesof epithelial or endocrine tissues including respiratory systemcarcinomas, gastrointestinal system carcinomas, genitourinary systemcarcinomas, testicular carcinomas, breast carcinomas, prostaticcarcinomas, endocrine system carcinomas, and melanomas. Exemplarycarcinomas include those forming from tissue of the cervix, lung,prostate, breast, head and neck, colon and ovary. The term also includescarcinosarcomas, e.g., which include malignant tumors composed ofcarcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to acarcinoma derived from glandular tissue or in which the tumor cells formrecognizable glandular structures.

[0039] The term “sarcoma” is art recognized and refers to malignanttumors of mesenchymal derivation.

[0040] The cytidine deaminase-like nucleic acid and protein of theinvention can be used to treat and/or diagnose a variety ofproliferative disorders. E.g., such disorders include hematopoieticneoplastic disorders. As used herein, the term “hematopoietic neoplasticdisorders” includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin, e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof. Preferably, the diseases arisefrom poorly differentiated acute leukemias, e.g., erythroblasticleukemia and acute megakaryoblastic leukemia. Additional exemplarymyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit. Rev. inOncol./Hemotol. 11:267-97); lymphoid malignancies include, but are notlimited to acute lymphoblastic leukemia (ALL) which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

[0041] The disclosed invention relates to methods and compositions forthe modulation, diagnosis, and treatment of a variety of disorders.Disorders involving the spleen include, but are not limited to,splenomegaly, including nonspecific acute splenitis, congestivespenomegaly, and spenic infarcts; neoplasms, congenital anomalies, andrupture. Disorders associated with splenomegaly include infections, suchas nonspecific splenitis, infectious mononucleosis, tuberculosis,typhoid fever, brucellosis, cytomegalovirus, syphilis, malaria,histoplasmosis, toxoplasmosis, kala-azar, trypanosomiasis,schistosomiasis, leishmaniasis, and echinococcosis; congestive statesrelated to partial hypertension, such as cirrhosis of the liver, portalor splenic vein thrombosis, and cardiac failure; lymphohematogenousdisorders, such as Hodgkin disease, non-Hodgkin lymphomas/leukemia,multiple myeloma, myeloproliferative disorders, hemolytic anemias, andthrombocytopenic purpura; immunologic-inflammatory conditions, such asrheumatoid arthritis and systemic lupus erythematosus; storage diseasessuch as Gaucher disease, Niemann-Pick disease, andmucopolysaccharidoses; and other conditions, such as amyloidosis,primary neoplasms and cysts, and secondary neoplasms.

[0042] Disorders involving the lung include, but are not limited to,congenital anomalies; atelectasis; diseases of vascular origin, such aspulmonary congestion and edema, including hemodynamic pulmonary edemaand edema caused by microvascular injury, adult respiratory distresssyndrome (diffuse alveolar damage), pulmonary embolism, hemorrhage, andinfarction, and pulmonary hypertension and vascular sclerosis; chronicobstructive pulmonary disease, such as emphysema, chronic bronchitis,bronchial asthma, and bronchiectasis; diffuse interstitial(infiltrative, restrictive) diseases, such as pneumoconioses,sarcoidosis, idiopathic pulmonary fibrosis, desquamative interstitialpneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia(pulmonary infiltration with eosinophilia), Bronchiolitisobliterans-organizing pneumonia, diffuse pulmonary hemorrhage syndromes,including Goodpasture syndrome, idiopathic pulmonary hemosiderosis andother hemorrhagic syndromes, pulmonary involvement in collagen vasculardisorders, and pulmonary alveolar proteinosis; complications oftherapies, such as drug-induced lung disease, radiation-induced lungdisease, and lung transplantation; tumors, such as bronchogeniccarcinoma, including paraneoplastic syndromes, bronchioloalveolarcarcinoma, neuroendocrine tumors, such as bronchial carcinoid,miscellaneous tumors, and metastatic tumors; pathologies of the pleura,including inflammatory pleural effusions, noninflammatory pleuraleffusions, pneumothorax, and pleural tumors, including solitary fibroustumors (pleural fibroma) and malignant mesothelioma.

[0043] Disorders involving the colon include, but are not limited to,congenital anomalies, such as atresia and stenosis, Meckel diverticulum,congenital aganglionic megacolon-Hirschsprung disease; enterocolitis,such as diarrhea and dysentery, infectious enterocolitis, includingviral gastroenteritis, bacterial enterocolitis, necrotizingenterocolitis, antibiotic-associated colitis (pseudomembranous colitis),and collagenous and lymphocytic colitis, miscellaneous intestinalinflammatory disorders, including parasites and protozoa, acquiredimmunodeficiency syndrome, transplantation, drug-induced intestinalinjury, radiation enterocolitis, neutropenic colitis (typhlitis), anddiversion colitis; idiopathic inflammatory bowel disease, such as Crohndisease and ulcerative colitis; tumors of the colon, such asnon-neoplastic polyps, adenomas, familial syndromes, colorectalcarcinogenesis, colorectal carcinoma, and carcinoid tumors.

[0044] Disorders involving the liver include, but are not limited to,hepatic injury; jaundice and cholestasis, such as bilirubin and bileformation; hepatic failure and cirrhosis, such as cirrhosis, portalhypertension, including ascites, portosystemic shunts, and splenomegaly;infectious disorders, such as viral hepatitis, including hepatitis A-Einfection and infection by other hepatitis viruses, clinicopathologicsyndromes, such as the carrier state, asymptomatic infection, acuteviral hepatitis, chronic viral hepatitis, and fulminant hepatitis;autoimmune hepatitis; drug- and toxin-induced liver disease, such asalcoholic liver disease; inborn errors of metabolism and pediatric liverdisease, such as hemochromatosis, Wilson disease, α₁-antitrypsindeficiency, and neonatal hepatitis; intrahepatic biliary tract disease,such as secondary biliary cirrhosis, primary biliary cirrhosis, primarysclerosing cholangitis, and anomalies of the biliary tree; circulatorydisorders, such as impaired blood flow into the liver, including hepaticartery compromise and portal vein obstruction and thrombosis, impairedblood flow through the liver, including passive congestion andcentrilobular necrosis and peliosis hepatis, hepatic vein outflowobstruction, including hepatic vein thrombosis (Budd-Chiari syndrome)and veno-occlusive disease; hepatic disease associated with pregnancy,such as preeclampsia and eclampsia, acute fatty liver of pregnancy, andintrehepatic cholestasis of pregnancy; hepatic complications of organ orbone marrow transplantation, such as drug toxicity after bone marrowtransplantation, graft-versus-host disease and liver rejection, andnonimmunologic damage to liver allografts; tumors and tumorousconditions, such as nodular hyperplasias, adenomas, and malignanttumors, including primary carcinoma of the liver and metastatic tumors.

[0045] Disorders involving the uterus and endometrium include, but arenot limited to, endometrial histology in the menstrual cycle; functionalendometrial disorders, such as anovulatory cycle, inadequate lutealphase, oral contraceptives and induced endometrial changes, andmenopausal and postmenopausal changes; inflammations, such as chronicendometritis; adenomyosis; endometriosis; endometrial polyps;endometrial hyperplasia; malignant tumors, such as carcinoma of theendometrium; mixed Müllerian and mesenchymal tumors, such as malignantmixed Müllerian tumors; tumors of the myometrium, including leiomyomas,leiomyosarcomas, and endometrial stromal tumors.

[0046] Disorders involving the brain include, but are not limited to,disorders involving neurons, and disorders involving glia, such asastrocytes, oligodendrocytes, ependymal cells, and microglia; cerebraledema, raised intracranial pressure and herniation, and hydrocephalus;malformations and developmental diseases, such as neural tube defects,forebrain anomalies, posterior fossa anomalies, and syringomyelia andhydromyelia; perinatal brain injury; cerebrovascular diseases, such asthose related to hypoxia, ischemia, and infarction, includinghypotension, hypoperfusion, and low-flow states—global cerebral ischemiaand focal cerebral ischemia—infarction from obstruction of local bloodsupply, intracranial hemorrhage, including intracerebral(intraparenchymal) hemorrhage, subarachnoid hemorrhage and rupturedberry aneurysms, and vascular malformations, hypertensivecerebrovascular disease, including lacunar infarcts, slit hemorrhages,and hypertensive encephalopathy; infections, such as acute meningitis,including acute pyogenic (bacterial) meningitis and acute aseptic(viral) meningitis, acute focal suppurative infections, including brainabscess, subdural empyema, and extradural abscess, chronic bacterialmeningoencephalitis, including tuberculosis and mycobacterioses,neurosyphilis, and neuroborreliosis (Lyme disease), viralmeningoencephalitis, including arthropod-borne (Arbo) viralencephalitis, Herpes simplex virus Type 1, Herpes simplex virus Type 2,Varicalla-zoster virus (Herpes zoster), cytomegalovirus, poliomyelitis,rabies, and human immunodeficiency virus 1, including HIV-1meningoencephalitis (subacute encephalitis), vacuolar myelopathy,AIDS-associated myopathy, peripheral neuropathy, and AIDS in children,progressive multifocal leukoencephalopathy, subacute sclerosingpanencephalitis, fungal meningoencephalitis, other infectious diseasesof the nervous system; transmissible spongiform encephalopathies (priondiseases); demyelinating diseases, including multiple sclerosis,multiple sclerosis variants, acute disseminated encephalomyelitis andacute necrotizing hemorrhagic encephalomyelitis, and other diseases withdemyelination; degenerative diseases, such as degenerative diseasesaffecting the cerebral cortex, including Alzheimer disease and Pickdisease, degenerative diseases of basal ganglia and brain stem,including Parkinsonism, idiopathic Parkinson disease (paralysisagitans), progressive supranuclear palsy, corticobasal degenration,multiple system atrophy, including striatonigral degenration, Shy-Dragersyndrome, and olivopontocerebellar atrophy, and Huntington disease;spinocerebellar degenerations, including spinocerebellar ataxias,including Friedreich ataxia, and ataxia-telanglectasia, degenerativediseases affecting motor neurons, including amyotrophic lateralsclerosis (motor neuron disease), bulbospinal atrophy (Kennedysyndrome), and spinal muscular atrophy; inborn errors of metabolism,such as leukodystrophies, including Krabbe disease, metachromaticleukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease, andCanavan disease, mitochondrial encephalomyopathies, including Leighdisease and other mitochondrial encephalomyopathies; toxic and acquiredmetabolic diseases, including vitamin deficiencies such as thiamine(vitamin B₁) deficiency and vitamin B₁₂ deficiency, neurologic sequelaeof metabolic disturbances, including hypoglycemia, hyperglycemia, andhepatic encephatopathy, toxic disorders, including carbon monoxide,methanol, ethanol, and radiation, including combined methotrexate andradiation-induced injury; tumors, such as gliomas, includingastrocytoma, including fibrillary (diffuse) astrocytoma and glioblastomamultiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, andbrain stem glioma, oligodendroglioma, and ependymoma and relatedparaventricular mass lesions, neuronal tumors, poorly differentiatedneoplasms, including medulloblastoma, other parenchymal tumors,including primary brain lymphoma, germ cell tumors, and pinealparenchymal tumors, meningiomas, metastatic tumors, paraneoplasticsyndromes, peripheral nerve sheath tumors, including schwannoma,neurofibroma, and malignant peripheral nerve sheath tumor (malignantschwannoma), and neurocutaneous syndromes (phakomatoses), includingneurofibromotosis, including Type 1 neurofibromatosis (NF1) and TYPE 2neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindaudisease.

[0047] Disorders involving T-cells include, but are not limited to,cell-mediated hypersensitivity, such as delayed type hypersensitivityand T-cell-mediated cytotoxicity, and transplant rejection; autoimmunediseases, such as systemic lupus erythematosus, Sjögren syndrome,systemic sclerosis, inflammatory myopathies, mixed connective tissuedisease, and polyarteritis nodosa and other vasculitides; immunologicdeficiency syndromes, including but not limited to, primaryimmunodeficiencies, such as thymic hypoplasia, severe combinedimmunodeficiency diseases, and AIDS; leukopenia; reactive (inflammatory)proliferations of white cells, including but not limited to,leukocytosis, acute nonspecific lymphadenitis, and chronic nonspecificlymphadenitis; neoplastic proliferations of white cells, including butnot limited to lymphoid neoplasms, such as precursor T-cell neoplasms,such as acute lymphoblastic leukemia/lymphoma, peripheral T-cell andnatural killer cell neoplasms that include peripheral T-cell lymphoma,unspecified, adult T-cell leukemia/lymphoma, mycosis fungoides andSézary syndrome, and Hodgkin disease.

[0048] Diseases of the skin, include but are not limited to, disordersof pigmentation and melanocytes, including but not limited to, vitiligo,freckle, melasma, lentigo, nevocellular nevus, dysplastic nevi, andmalignant melanoma; benign epithelial tumors, including but not limitedto, seborrheic keratoses, acanthosis nigricans, fibroepithelial polyp,epithelial cyst, keratoacanthoma, and adnexal (appendage) tumors;premalignant and malignant epidermal tumors, including but not limitedto, actinic keratosis, squamous cell carcinoma, basal cell carcinoma,and merkel cell carcinoma; tumors of the dermis, including but notlimited to, benign fibrous histiocytoma, dermatofibrosarcomaprotuberans, xanthomas, and dermal vascular tumors; tumors of cellularimmigrants to the skin, including but not limited to, histiocytosis X,mycosis fungoides (cutaneous T-cell lymphoma), and mastocytosis;disorders of epidermal maturation, including but not limited to,ichthyosis; acute inflammatory dermatoses, including but not limited to,urticaria, acute eczematous dermatitis, and erythema multiforme; chronicinflammatory dermatoses, including but not limited to, psoriasis, lichenplanus, and lupus erythematosus; blistering (bullous) diseases,including but not limited to, pemphigus, bullous pemphigoid, dermatitisherpetiformis, and noninflammatory blistering diseases: epidermolysisbullosa and porphyria; disorders of epidermal appendages, including butnot limited to, acne vulgaris; panniculitis, including but not limitedto, erythema nodosum and erythema induratum; and infection andinfestation, such as verrucae, molluscum contagiosum, impetigo,superficial fungal infections, and arthropod bites, stings, andinfestations.

[0049] In normal bone marrow, the myelocytic series (polymorphoneuclearcells) make up approximately 60% of the cellular elements, and theerythrocytic series, 20-30%. Lymphocytes, monocytes, reticular cells,plasma cells and megakaryocytes together constitute 10-20%. Lymphocytesmake up 5-15% of normal adult marrow. In the bone marrow, cell types areadd mixed so that precursors of red blood cells (erythroblasts),macrophages (monoblasts), platelets (megakaryocytes), polymorphoneuclearleucocytes (myeloblasts), and lymphocytes (lymphoblasts) can be visiblein one microscopic field. In addition, stem cells exist for thedifferent cell lineages, as well as a precursor stem cell for thecommitted progenitor cells of the different lineages. The various typesof cells and stages of each would be known to the person of ordinaryskill in the art and are found, for example, on page 42 (FIGS. 2-8) ofImmunology, Imunopathology and Immunity, Fifth Edition, Sell et al.Simon and Schuster (1996), incorporated by reference for its teaching ofcell types found in the bone marrow. According, the invention isdirected to disorders arising from these cells. These disorders includebut are not limited to the following: diseases involving hematopoeiticstem cells; committed lymphoid progenitor cells; lymphoid cellsincluding B and T-cells; committed myeloid progenitors, includingmonocytes, granulocytes, and megakaryocytes; and committed erythroidprogenitors. These include but are not limited to the leukemias,including B-lymphoid leukemias, T-lymphoid leukemias, undifferentiatedleukemias; erythroleukemia, megakaryoblastic leukemia, monocytic;[leukemias are encompassed with and without differentiation]; chronicand acute lymphoblastic leukemia, chronic and acute lymphocyticleukemia, chronic and acute myelogenous leukemia, lymphoma, myelodysplastic syndrome, chronic and acute myeloid leukemia, myelomonocyticleukemia; chronic and acute myeloblastic leukemia, chronic and acutemyelogenous leukemia, chronic and acute promyelocytic leukemia, chronicand acute myelocytic leukemia, hematologic malignancies ofmonocyte-macrophage lineage, such as juvenile chronic myelogenousleukemia; secondary AML, antecedent hematological disorder; refractoryanemia; aplastic anemia; reactive cutaneous angioendotheliomatosis;fibrosing disorders involving altered expression in dendritic cells,disorders including systemic sclerosis, E-M syndrome, epidemic toxic oilsyndrome, eosinophilic fasciitis localized forms of scleroderma, keloid,and fibrosing colonopathy; angiomatoid malignant fibrous histiocytoma;carcinoma, including primary head and neck squamous cell carcinoma;sarcoma, including kaposi's sarcoma; fibroadanoma and phyllodes tumors,including mammary fibroadenoma; stromal tumors; phyllodes tumors,including histiocytoma; erythroblastosis; neurofibromatosis; diseases ofthe vascular endothelium; demyelinating, particularly in old lesions;gliosis, vasogenic edema, vascular disease, Alzheimer's and Parkinson'sdisease; T-cell lymphomas; B-cell lymphomas.

[0050] Disorders involving the heart, include but are not limited to,heart failure, including but not limited to, cardiac hypertrophy,left-sided heart failure, and right-sided heart failure; ischemic heartdisease, including but not limited to angina pectoris, myocardialinfarction, chronic ischemic heart disease, and sudden cardiac death;hypertensive heart disease, including but not limited to, systemic(left-sided) hypertensive heart disease and pulmonary (right-sided)hypertensive heart disease; valvular heart disease, including but notlimited to, valvular degeneration caused by calcification, such ascalcific aortic stenosis, calcification of a congenitally bicuspidaortic valve, and mitral annular calcification, and myxomatousdegeneration of the mitral valve (mitral valve prolapse), rheumaticfever and rheumatic heart disease, infective endocarditis, andnoninfected vegetations, such as nonbacterial thrombotic endocarditisand endocarditis of systemic lupus erythematosus (Libman-Sacks disease),carcinoid heart disease, and complications of artificial valves;myocardial disease, including but not limited to dilated cardiomyopathy,hypertrophic cardiomyopathy, restrictive cardiomyopathy, andmyocarditis; pericardial disease, including but not limited to,pericardial effusion and hemopericardium and pericarditis, includingacute pericarditis and healed pericarditis, and rheumatoid heartdisease; neoplastic heart disease, including but not limited to, primarycardiac tumors, such as myxoma, lipoma, papillary fibroelastoma,rhabdomyoma, and sarcoma, and cardiac effects of noncardiac neoplasms;congenital heart disease, including but not limited to, left-to-rightshunts—late cyanosis, such as atrial septal defect, ventricular septaldefect, patent ductus arteriosus, and atrioventricular septal defect,right-to-left shunts—early cyanosis, such as tetralogy of fallot,transposition of great arteries, truncus arteriosus, tricuspid atresia,and total anomalous pulmonary venous connection, obstructive congenitalanomalies, such as coarctation of aorta, pulmonary stenosis and atresia,and aortic stenosis and atresia, and disorders involving cardiactransplantation.

[0051] Disorders involving blood vessels include, but are not limitedto, responses of vascular cell walls to injury, such as endothelialdysfunction and endothelial activation and intimal thickening; vasculardiseases including, but not limited to, congenital anomalies, such asarteriovenous fistula, atherosclerosis, and hypertensive vasculardisease, such as hypertension; inflammatory disease—the vasculitides,such as giant cell (temporal) arteritis, Takayasu arteritis,polyarteritis nodosa (classic), Kawasaki syndrome (mucocutaneous lymphnode syndrome), microscopic polyanglitis (microscopic polyarteritis,hypersensitivity or leukocytoclastic anglitis), Wegener granulomatosis,thromboanglitis obliterans (Buerger disease), vasculitis associated withother disorders, and infectious arteritis; Raynaud disease; aneurysmsand dissection, such as abdominal aortic aneurysms, syphilitic (luetic)aneurysms, and aortic dissection (dissecting hematoma); disorders ofveins and lymphatics, such as varicose veins, thrombophlebitis andphlebothrombosis, obstruction of superior vena cava (superior vena cavasyndrome), obstruction of inferior vena cava (inferior vena cavasyndrome), and lymphangitis and lymphedema; tumors, including benigntumors and tumor-like conditions, such as hemangioma, lymphangioma,glomus tumor (glomangioma), vascular ectasias, and bacillaryangiomatosis, and intermediate-grade (borderline low-grade malignant)tumors, such as Kaposi sarcoma and hemangloendothelioma, and malignanttumors, such as angiosarcoma and hemangiopericytoma; and pathology oftherapeutic interventions in vascular disease, such as balloonangioplasty and related techniques and vascular replacement, such ascoronary artery bypass graft surgery.

[0052] Disorders involving red cells include, but are not limited to,anemias, such as hemolytic anemias, including hereditary spherocytosis,hemolytic disease due to erythrocyte enzyme defects: glucose-6-phosphatedehydrogenase deficiency, sickle cell disease, thalassemia syndromes,paroxysmal nocturnal hemoglobinuria, immunohemolytic anemia, andhemolytic anemia resulting from trauma to red cells; and anemias ofdiminished erythropoiesis, including megaloblastic anemias, such asanemias of vitamin B12 deficiency: pernicious anemia, and anemia offolate deficiency, iron deficiency anemia, anemia of chronic disease,aplastic anemia, pure red cell aplasia, and other forms of marrowfailure.

[0053] Disorders involving the thymus include developmental disorders,such as DiGeorge syndrome with thymic hypoplasia or aplasia; thymiccysts; thymic hypoplasia, which involves the appearance of lymphoidfollicles within the thymus, creating thymic follicular hyperplasia; andthymomas, including germ cell tumors, lynphomas, Hodgkin disease, andcarcinoids. Thymomas can include benign or encapsulated thymoma, andmalignant thymoma Type I (invasive thymoma) or Type II, designatedthymic carcinoma.

[0054] Disorders involving B-cells include, but are not limited toprecursor B-cell neoplasms, such as lymphoblastic leukemiaAymphoma.Peripheral B-cell neoplasms include, but are not limited to, chroniclymphocytic leukemia/small lymphocytic lymphoma, follicular lymphoma,diffuse large B-cell lymphoma, Burkitt lymphoma, plasma cell neoplasms,multiple myeloma, and related entities, lymphoplasmacytic lymphoma(Waldenstr{overscore (o)}m macroglobulinemia), mantle cell lymphoma,marginal zone lymphoma (MALToma), and hairy cell leukemia.

[0055] Disorders involving the kidney include, but are not limited to,congenital anomalies including, but not limited to, cystic diseases ofthe kidney, that include but are not limited to, cystic renal dysplasia,autosomal dominant (adult) polycystic kidney disease, autosomalrecessive (childhood) polycystic kidney disease, and cystic diseases ofrenal medulla, which include, but are not limited to, medullary spongekidney, and nephronophthisis-uremic medullary cystic disease complex,acquired (dialysis-associated) cystic disease, such as simple cysts;glomerular diseases including pathologies of glomerular injury thatinclude, but are not limited to, in situ immune complex deposition, thatincludes, but is not limited to, anti-GBM nephritis, Heymann nephritis,and antibodies against planted antigens, circulating immune complexnephritis, antibodies to glomerular cells, cell-mediated immunity inglomerulonephritis, activation of alternative complement pathway,epithelial cell injury, and pathologies involving mediators ofglomerular injury including cellular and soluble mediators, acuteglomerulonephritis, such as acute proliferative (poststreptococcal,postinfectious) glomerulonephritis, including but not limited to,poststreptococcal glomerulonephritis and nonstreptococcal acuteglomerulonephritis, rapidly progressive (crescentic) glomerulonephritis,nephrotic syndrome, membranous glomerulonephritis (membranousnephropathy), minimal change disease (lipoid nephrosis), focal segmentalglomerulosclerosis, membranoproliferative glomerulonephritis, IgAnephropathy (Berger disease), focal proliferative and necrotizingglomerulonephritis (focal glomerulonephritis), hereditary nephritis,including but not limited to, Alport syndrome and thin membrane disease(benign familial hematuria), chronic glomerulonephritis, glomerularlesions associated with systemic disease, including but not limited to,systemic lupus erythematosus, Henoch-Schönlein purpura, bacterialendocarditis, diabetic glomerulosclerosis, amyloidosis, fibrillary andimmunotactoid glomerulonephritis, and other systemic disorders; diseasesaffecting tubules and interstitium, including acute tubular necrosis andtubulointerstitial nephritis, including but not limited to,pyelonephritis and urinary tract infection, acute pyelonephritis,chronic pyelonephritis and reflux nephropathy, and tubulointerstitialnephritis induced by drugs and toxins, including but not limited to,acute drug-induced interstitial nephritis, analgesic abuse nephropathy,nephropathy associated with nonsteroidal anti-inflammatory drugs, andother tubulointerstitial diseases including, but not limited to, uratenephropathy, hypercalcemia and nephrocalcinosis, and multiple myeloma;diseases of blood vessels including benign nephrosclerosis, malignanthypertension and accelerated nephroscierosis, renal artery stenosis, andthrombotic microangiopathies including, but not limited to, classic(childhood) hemolytic-uremic syndrome, adult hemolytic-uremicsyndrome/thrombotic thrombocytopenic purpura, idiopathic HUS/TTP, andother vascular disorders including, but not limited to, atheroscleroticischemic renal disease, atheroembolic renal disease, sickle cell diseasenephropathy, diffuse cortical necrosis, and renal infarcts; urinarytract obstruction (obstructive uropathy); urolithiasis (renal calculi,stones); and tumors of the kidney including, but not limited to, benigntumors, such as renal papillary adenoma, renal fibroma or hamartoma(renomedullary interstitial cell tumor), angiomyolipoma, and oncocytoma,and malignant tumors, including renal cell carcinoma (hypernephroma,adenocarcinoma of kidney), which includes urothelial carcinomas of renalpelvis.

[0056] Disorders of the breast include, but are not limited to,disorders of development; inflammations, including but not limited to,acute mastitis, periductal mastitis, periductal mastitis (recurrentsubareolar abscess, squamous metaplasia of lactiferous ducts), mammaryduct ectasia, fat necrosis, granulomatous mastitis, and pathologiesassociated with silicone breast implants; fibrocystic changes;proliferative breast disease including, but not limited to, epithelialhyperplasia, sclerosing adenosis, and small duct papillomas; tumorsincluding, but not limited to, stromal tumors such as fibroadenoma,phyllodes tumor, and sarcomas, and epithelial tumors such as large ductpapilloma; carcinoma of the breast including in situ (noninvasive)carcinoma that includes ductal carcinoma in situ (including Paget'sdisease) and lobular carcinoma in situ, and invasive (infiltrating)carcinoma including, but not limited to, invasive ductal carcinoma, nospecial type, invasive lobular carcinoma, medullary carcinoma, colloid(mucinous) carcinoma, tubular carcinoma, and invasive papillarycarcinoma, and miscellaneous malignant neoplasms.

[0057] Disorders in the male breast include, but are not limited to,gynecomastia and carcinoma.

[0058] Disorders involving the testis and epididymis include, but arenot limited to, congenital anomalies such as cryptorchidism, regressivechanges such as atrophy, inflammations such as nonspecific epididymitisand orchitis, granulomatous (autoimmune) orchitis, and specificinflammations including, but not limited to, gonorrhea, mumps,tuberculosis, and syphilis, vascular disturbances including torsion,testicular tumors including germ cell tumors that include, but are notlimited to, seminoma, spermatocytic seminoma, embryonal carcinoma, yolksac tumor choriocarcinoma, teratoma, and mixed tumors, tumore of sexcord-gonadal stroma including, but not limited to, Leydig (interstitial)cell tumors and sertoli cell tumors (androblastoma), and testicularlymphoma, and miscellaneous lesions of tunica vaginalis.

[0059] Disorders involving the prostate include, but are not limited to,inflammations, benign enlargement, for example, nodular hyperplasia(benign prostatic hypertrophy or hyperplasia), and tumors such ascarcinoma.

[0060] Disorders involving the thyroid include, but are not limited to,hyperthyroidism; hypothyroidism including, but not limited to, cretinismand myxedema; thyroiditis including, but not limited to, hashimotothyroiditis, subacute (granulomatous) thyroiditis, and subacutelymphocytic (painless) thyroiditis; Graves disease; diffuse andmultinodular goiter including, but not limited to, diffuse nontoxic(simple) goiter and multinodular goiter; neoplasms of the thyroidincluding, but not limited to, adenomas, other benign tumors, andcarcinomas, which include, but are not limited to, papillary carcinoma,follicular carcinoma, medullary carcinoma, and anaplastic carcinoma; andcogenital anomalies.

[0061] Disorders involving the skeletal muscle include tumors such asrhabdomyosarcoma.

[0062] Disorders involving the pancreas include those of the exocrinepancreas such as congenital anomalies, including but not limited to,ectopic pancreas; pancreatitis, including but not limited to, acutepancreatitis; cysts, including but not limited to, pseudocysts; tumors,including but not limited to, cystic tumors and carcinoma of thepancreas; and disorders of the endocrine pancreas such as, diabetesmellitus; islet cell tumors, including but not limited to, insulinomas,gastrinomas, and other rare islet cell tumors.

[0063] Disorders involving the small intestine include the malabsorptionsyndromes such as, celiac sprue, tropical sprue (postinfectious sprue),whipple disease, disaccharidase (lactase) deficiency,abetalipoproteinemia, and tumors of the small intestine includingadenomas and adenocarcinoma.

[0064] Disorders related to reduced platelet number, thrombocytopenia,include idiopathic thrombocytopenic purpura, including acute idiopathicthrombocytopenic purpura, drug-induced thrombocytopenia, HIV-associatedthrombocytopenia, and thrombotic microangiopathies: thromboticthrombocytopenic purpura and hemolytic-uremic syndrome.

[0065] Disorders involving precursor T-cell neoplasms include precursorT lymphoblastic leukemia/lymphoma. Disorders involving peripheral T-celland natural killer cell neoplasms include T-cell chronic lymphocyticleukemia, large granular lymphocytic leukemia, mycosis fungoides andSëzary syndrome, peripheral T-cell lymphoma, unspecified,angioimmunoblastic T-cell lymphoma, angiocentric lymphoma (NK/T-celllymphoma^(4a)), intestinal T-cell lymphoma, adult T-cellleukemia/lymphoma, and anaplastic large cell lymphoma.

[0066] Disorders involving the ovary include, for example, polycysticovarian disease, Stein-leventhal syndrome, Pseudomyxoma peritonei andstromal hyperthecosis; ovarian tumors such as, tumors of coelomicepithelium, serous tumors, mucinous tumors, endometeriod tumors, clearcell adenocarcinoma, cystadenofibroma, brenner tumor, surface epithelialtumors; germ cell tumors such as mature (benign) teratomas, monodermalteratomas, immature malignant teratomas, dysgerminoma, endodermal sinustumor, choriocarcinoma; sex cord-stomal tumors such as, granulosa-thecacell tumors, thecoma-fibromas, androblastomas, hill cell tumors, andgonadoblastoma; and metastatic tumors such as Krukenberg tumors.

[0067] Bone-forming cells include the osteoprogenitor cells,osteoblasts, and osteocytes. The disorders of the bone are complexbecause they may have an impact on the skeleton during any of its stagesof development. Hence, the disorders may have variable manifestationsand may involve one, multiple or all bones of the body. Such disordersinclude, congenital malformations, achondroplasia and thanatophoricdwarfism, diseases associated with abnormal matix such as type 1collagen disease, osteoporosis, Paget disease, rickets, osteomalacia,high-turnover, osteodystrophy, low-turnover of aplastic disease,osteonecrosis, pyogenic osteomyelitis, tuberculous osteomyelitism,osteoma, osteoid osteoma, osteoblastoma, osteosarcoma, osteochondroma,chondromas, chondroblastoma, chondromyxoid fibroma, chondrosarcoma,fibrous cortical defects, fibrous dysplasia, fibrosarcoma, malignantfibrous histiocytoma, Ewing sarcoma, primitive neuroectodermal tumor,giant cell tumor, and metastatic tumors.

[0068] The cytidine deaminase-like gene, clone 26934, was identified ina primary osteoblast cDNA library. Clone 26934 encodes an mRNAtranscript having the corresponding cDNA set forth in SEQ ID NO:1. Thistranscript has a 1020 nucleotide open reading frame (nucleotides147-1,167 of SEQ ID NO:1), which encodes a 339 amino acid protein (SEQID NO:2). An analysis of the full-length 26934 polypeptide predicts thatthe N-terminal 54 amino acids represent a signal peptide. Atransmembrane segment from amino acids (aa) 279-299 was predicted byMEMSAT. Transmembrane segments were also predicted from aa 133-150 andfrom 226-243 of the presumed mature peptide sequence. Prosite programanalysis was used to predict various sites within the 26934 protein. AnN-glycosylation site was predicted at aa 311-314. Protein kinase Cphosphorylation sites were predicted at aa 12-14, 58-60, 80-82, 130-132,and 207-209. Casein kinase II phosphorylation sites were predicted at aa104-107, 165-168, 219-222, 246-249, and 301-304. N-myristoylation siteswere predicted at aa 5-10, 24-29, and 100-105. A leucine zipper motifwas predicted at aa 101-122. The cytidine deaminase-like proteinpossesses a cytidine and deoxycytidylate deaminase zinc-binding region,from aa 80-149, as predicted by HMMer, Version 2. For generalinformation regarding PFAM identifiers, PS prefix and PF prefix domainidentification numbers, refer to Sonnhammer et al. (1997) Protein28:405-420 andhttp//www.psc.edu/general/software/packages/pfam/pfam.html. Cytidinedeaminase (EC 3.5.4.5) catalyzes the hydrolysis of cytidine into uridineand ammonia while deoxycytidylate deaminase (EC 3.5.4.12) hydrolyzesdCMP into dUMP. Both enzymes are known to bind zinc and to require itfor their catalytic activity. These two enzymes contain a region ofthree conserved histidine and cysteine residues which are thought to beinvolved in the binding of the catalytic zinc ion. See for example,Bhattacharya et al. (1994) Trends in Biochem. Sci. 19:105-106 and Reizeret al. (1994) Protein Sci. 3:853-856. The cytidine deaminase-likeprotein also possesses a Hint (Hedgehog/Intein) domain C-terminal regionfrom aa 71-91, as predicted by HMMer, Version 2 using the SMARTdatabase.

[0069] As used herein, the term “cytidine deoxycytidylate deaminasezinc-binding region domain” includes an amino acid sequence of about1-69 amino acid residues in length and having a bit score for thealignment of the sequence to the cytidine deoxycytidylate deaminasezinc-binding region domain (HMM) of at least 8. Preferably, an cytidinedeoxycytidylate deaminase zinc-binding region domain includes at leastabout 1 to 69 amino acids, more about 1 to 25 amino acid residues, orabout 25-60 amino acids and has a bit score for the alignment of thesequence to the cytidine deoxycytidylate deaminase zinc-binding regiondomain (HMM) of at least 16 or greater. The cytidine deoxycytidylatedeaminase zinc-binding region domain (HMM) has been assigned the PFAMAccession PDOC00702 (http;//pfam.wustl.edu/). An alignment of thecytidine deoxycytidylate deaminase zinc-binding region domain (aminoacids 80 to 149 of SEQ ID NO:2) of human cytidine deaminase-likemolecule of the invention with a consensus amino acid sequence derivedfrom a hidden Markov model is depicted in FIG. 3.

[0070] In a preferred embodiment cytidine deaminase-like polypeptide orprotein has a “cytidine deoxycytidylate deaminase zinc-binding regiondomain” or a region which includes at least about 100-250 morepreferably about 130-200 or 160-200 amino acid residues and has at leastabout 60%, 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with an“cytidine deoxycytidylate deaminase zinc-binding region domain,” e.g.,the cytidine deoxycytidylate deaminase zinc-binding region domain ofhuman the cytidine deaminase-like (e.g., amino acid residues 80-149 ofSEQ ID NO:2).

[0071] To identify the presence of an “cytidine deoxycytidylatedeaminase zinc-binding region” domain in a cytidine deaminase-likeprotein sequence, and make the determination that a polypeptide orprotein of interest has a particular profile, the amino acid sequence ofthe protein can be searched against a database of HMMs (e.g., the Pfamdatabase, release 2.1) using the default parameters(http://www.sanger.ac.uk/Software/Pfam/HMM_search). For example, thehmmsf program, which is available as part of the HMMER package of searchprograms, is a family specific default program for MILPAT0063 and ascore of 15 is the default threshold score for determining a hit.Alternatively, the threshold score for determining a hit can be lowered(e.g., to 8 bits). A description of the Pfam database can be found inSonhammer et al. (1997) Proteins 28(3):405-420 and a detaileddescription of HMMs can be found, for example, in Gribskov et al. (1990)Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad.Sci. USA 84:43554358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531;and Stultz et al. (1993) Protein Sci. 2:305-314, the contents of whichare incorporated herein by reference.

[0072] In one embodiment, a cytidine deaminase-like protein includes atleast one transmembrane domain. As used herein, the term “transmembranedomain” includes an amino acid sequence of about 15 amino acid residuesin length that spans a phospholipid membrane. More preferably, atransmembrane domain includes about at least 18, 20, 22, 24, 25, 30, 35or 40 amino acid residues and spans a phospholipid membrane.Transmembrane domains are rich in hydrophobic residues, and typicallyhave an α-helical structure. In a preferred embodiment, at least 50%,60%, 70%, 80%, 90%, 95% or more of the amino acids of a transmembranedomain are hydrophobic, e.g., leucines, isoleucines, tyrosines, ortryptophans. Transmembrane domains are described in, for example,http://pfam.wustl.edu/cgi-bin/getdesc?name=7tm-1, and Zagotta W. N. etal. (1996) Annual Rev. Neuronsci. 19:235-63, the contents of which areincorporated herein by reference.

[0073] In a preferred embodiment, a cytidine deaminase-like polypeptideor protein has at least one transmembrane domain or a region whichincludes at least 18, 20, 22, 24, 25, 30, 35 or 40 amino acid residuesand has at least about 60%, 70% 80% 90% 95%, 99%, or 100% sequenceidentity with a “transmembrane domain,” e.g., at least one transmembranedomain of human cytidine deaminase-like (e.g., amino acid residues279-299 of SEQ ID NO:2).

[0074] In another embodiment, a cytidine deaminase-like protein includesat least one “non-transmembrane domain.” As used herein,“non-transmembrane domains” are domains that reside outside of themembrane. When referring to plasma membranes, non-transmembrane domainsinclude extracellular domains (i.e., outside of the cell) andintracellular domains (i.e., within the cell). When referring tomembrane-bound proteins found in intracellular organelles (e.g.,mitochondria, endoplasmic reticulum, peroxisomes and microsomes),non-transmembrane domains include those domains of the protein thatreside in the cytosol (i.e., the cytoplasm), the lumen of the organelle,or the matrix or the intermembrane space (the latter two relatespecifically to mitochondria organelles). The C-terminal amino acidresidue of a non-transmembrane domain is adjacent to an N-terminal aminoacid residue of a transmembrane domain in a naturally occurring cytidinedeaminase-like or cytidine deaminase-like protein.

[0075] In a preferred embodiment, a cytidine deaminase-like polypeptideor protein has a “non-transmembrane domain” or a region which includesat least about 1-279, about 100-200, about 150-250, and about 50-175amino acid residues, and has at least about 60%, 70% 80% 90% 95%, 99% or100% sequence identity with a “non-transmembrane domain”, e.g., anon-transmembrane domain of human cytidine deaminase-like (e.g.,residues 1-279 and 300-340 of SEQ ID NO:2). Preferably, anon-transmembrane domain is capable of catalytic activity (e.g.,cytidine deaminase-like activity).

[0076] A non-transmembrane domain located at the N-terminus of acytidine deaminase-like protein or polypeptide is referred to herein asan “N-terminal non-transmembrane domain.” As used herein, an “N-terminalnon-transmembrane domain” includes an amino acid sequence having about1-350, preferably about 30-325, more preferably about 50-320, or evenmore preferably about 80-310 amino acid residues in length and islocated outside the boundaries of a membrane. For example, an N-terminalnon-transmembrane domain is located at about amino acid residues 1-279of SEQ ID NO:2.

[0077] Similarly, a non-transmembrane domain located at the C-terminusof a cytidine deaminase-like protein or polypeptide is referred toherein as a “C-terminal non-transmembrane domain.” As used herein, an“C-terminal non-transmembrane domain” includes an amino acid sequencehaving about 1-300, preferably about 15-290, preferably about 20-270,more preferably about 25-255 amino acid residues in length and islocated outside the boundaries of a membrane. For example, an C-terminalnon-transmembrane domain is located at about amino acid residues 300-340of SEQ ID NO:2.

[0078] A cytidine deaminase-like molecule can further include a signalsequence. As used herein, a “signal sequence” refers to a peptide ofabout 20-80 amino acid residues in length which occurs at the N-terminusof secretory and integral membrane proteins and which contains amajority of hydrophobic amino acid residues. For example, a signalsequence contains at least about 12-25 amino acid residues, preferablyabout 30-70 amino acid residues, more preferably about 61 amino acidresidues, and has at least about 40-70%, preferably about 50-65%, andmore preferably about 55-60% hydrophobic amino acid residues (e.g.,alanine, valine, leucine, isoleucine, phenylalanine, tyrosine,tryptophan, or proline). Such a “signal sequence”, also referred to inthe art as a “signal peptide”, serves to direct a protein containingsuch a sequence to a lipid bilayer. For example, in one embodiment, acytidine deaminase-like protein contains a signal sequence of aboutamino acids 1-54 of SEQ ID NO:2. The “signal sequence” is cleaved duringprocessing of the mature protein. The mature cytidine deaminase-likeprotein corresponds to amino acids 55-339 of SEQ ID NO:2.

[0079] The cytidine deaminase-like sequences of the invention aremembers of a family of molecules (the “cytidine deaminase”) havingconserved functional features. The term “family” when referring to theproteins and nucleic acid molecules of the invention is intended to meantwo or more proteins or nucleic acid molecules having sufficient aminoacid or nucleotide sequence identity as defined herein. Such familymembers can be naturally occurring and can be from either the same ordifferent species. For example, a family can contain a first protein ofmurine origin and a homologue of that protein of human origin, as wellas a second, distinct protein of human origin and a murine homologue ofthat protein. Members of a family may also have common functionalcharacteristics.

[0080] Preferred cytidine deaminase-like polypeptides of the presentinvention have an amino acid sequence sufficiently identical to theamino acid sequence of SEQ ID NO:2. The term “sufficiently identical” isused herein to refer to a first amino acid or nucleotide sequence thatcontains a sufficient or minimum number of identical or equivalent(e.g., with a similar side chain) amino acid residues or nucleotides toa second amino acid or nucleotide sequence such that the first andsecond amino acid or nucleotide sequences have a common structuraldomain and/or common functional activity. For example, amino acid ornucleotide sequences that contain a common structural domain having atleast about 45%, 55%, or 65% identity, preferably 75% identity, morepreferably 85%, 95%, or 98% identity are defined herein as sufficientlyidentical.

[0081] To determine the percent identity of two amino acid sequences orof two nucleic acids, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. The percent identity between two sequences can bedetermined using techniques similar to those described below, with orwithout allowing gaps. In calculating percent identity, typically exactmatches are counted.

[0082] The determination of percent identity between two sequences canbe accomplished using a mathematical algorithm. In a preferredembodiment, the percent identity between two amino acid sequences isdetermined using the Needleman and Wunsch (1970) J. Mol. Biol.48:444-453 algorithm which has been incorporated into the GAP program inthe GCG software package (available at http://www.gcg.com), using eithera Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12,10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yetanother preferred embodiment, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package (available at http://www.gcg.com), using aNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and alength weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set ofparameters (and the one that should be used if the practitioner isuncertain about what parameters should be applied to determine if amolecule is within a sequence identity or homology limitation of theinvention) is using a Blossum 62 scoring matrix with a gap open penaltyof 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0083] The percent identity between two amino acid or nucleotidesequences can be determined using the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al. (1990) J. Mol. Biol. 215:403. BLAST nucleotide searchescan be performed with the NBLAST program, score=100, wordlength=12, toobtain nucleotide sequences homologous to cytidine deaminase-likenucleic acid molecules of the invention. BLAST protein searches can beperformed with the XBLAST program, score=50, wordlength=3, to obtainamino acid sequences homologous to cytidine deaminase-like proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be usedto perform an iterated search that detects distant relationships betweenmolecules. See Altschul et al. (1997) supra. When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example ofa mathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an algorithmis incorporated into the ALIGN program (version 2.0), which is part ofthe GCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0084] Accordingly, another embodiment of the invention featuresisolated cytidine deaminase-like proteins and polypeptides having ancytidine deaminase-like protein activity. As used interchangeablyherein, a “cytidine deaminase-like protein activity”, “biologicalactivity of an cytidine deaminase-like protein”, or “functional activityof an cytidine deaminase-like protein” refers to an activity exerted byan cytidine deaminase-like protein, polypeptide, or nucleic acidmolecule on an cytidine deaminase-like responsive cell as determined invivo, or in vitro, according to standard assay techniques. A cytidinedeaminase-like activity can be a direct activity, such as an associationwith or an enzymatic activity on a second protein, or an indirectactivity, such as a cellular signaling activity mediated by interactionof the cytidine deaminase-like protein with a second protein. In apreferred embodiment, a cytidine deaminase-like activity includes atleast one or more of the following activities: (1) modulating(stimulating and/or enhancing or inhibiting) the conversion of cytidineand deoxycytidine to uridine and deoxyuridine, respectfully; (2)modulating an mRNA editing event; and (3) modulating the toxicity ofcytosine nucleoside analogs.

[0085] An “isolated” or “purified” cytidine deaminase-like nucleic acidmolecule or protein, or biologically active portion thereof, issubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. Preferably,an “isolated” nucleic acid is free of sequences (preferably proteinencoding sequences) that naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forpurposes of the invention, “isolated” when used to refer to nucleic acidmolecules excludes isolated chromosomes. For example, in variousembodiments, the isolated cytidine deaminase-like nucleic acid moleculecan contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1kb of nucleotide sequences that naturally flank the nucleic acidmolecule in genomic DNA of the cell from which the nucleic acid isderived. An cytidine deaminase-like protein that is substantially freeof cellular material includes preparations of cytidine deaminase-likeprotein having less than about 30%, 20%, 10%, or 5% (by dry weight) ofnon-cytidine deaminase-like protein (also referred to herein as a“contaminating protein”). When the cytidine deaminase-like protein orbiologically active portion thereof is recombinantly produced,preferably, culture medium represents less than about 30%, 20%, 10%, or5% of the volume of the protein preparation. When cytidinedeaminase-like protein is produced by chemical synthesis, preferably theprotein preparations have less than about 30%, 20%, 10%, or 5% (by dryweight) of chemical precursors or non-cytidine deaminase-like chemicals.

[0086] Various aspects of the invention are described in further detailin the following subsections.

[0087] I. Isolated Nucleic Acid Molecules

[0088] One aspect of the invention pertains to isolated nucleic acidmolecules comprising nucleotide sequences encoding cytidinedeaminase-like proteins and polypeptides or biologically active portionsthereof, as well as nucleic acid molecules sufficient for use ashybridization probes to identify cytidine deaminase-like-encodingnucleic acids (e.g., cytidine deaminase-like mRNA) and fragments for useas PCR primers for the amplification or mutation of cytidinedeaminase-like nucleic acid molecules. As used herein, the term “nucleicacid molecule” is intended to include DNA molecules (e.g., cDNA orgenomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

[0089] Nucleotide sequences encoding the cytidine deaminase-likeproteins of the present invention include sequences set forth in SEQ IDNO:1 or 3 and complements thereof. By “complement” is intended anucleotide sequence that is sufficiently complementary to a givennucleotide sequence such that it can hybridize to the given nucleotidesequence to thereby form a stable duplex. The corresponding amino acidsequence for the cytidine deaminase-like protein encoded by thesenucleotide sequences is set forth in SEQ ID NO:2. The invention alsoencompasses nucleic acid molecules comprising nucleotide sequencesencoding partial-length cytidine deaminase-like proteins, including thesequence set forth in SEQ ID NO:1 or 3, and complements thereof.

[0090] Nucleic acid molecules that are fragments of these cytidinedeaminase-like nucleotide sequences are also encompassed by the presentinvention. By “fragment” is intended a portion of the nucleotidesequence encoding an cytidine deaminase-like protein. A fragment of ancytidine deaminase-like nucleotide sequence may encode a biologicallyactive portion of an cytidine deaminase-like protein, or it may be afragment that can be used as a hybridization probe or PCR primer usingmethods disclosed below. A biologically active portion of an cytidinedeaminase-like protein can be prepared by isolating a portion of one ofthe 26934 nucleotide sequences of the invention, expressing the encodedportion of the cytidine deaminase-like protein (e.g., by recombinantexpression in vitro), and assessing the activity of the encoded portionof the cytidine deaminase-like protein. Nucleic acid molecules that arefragments of an cytidine deaminase-like nucleotide sequence comprise atleast about 15, 20, 50, 75, 100, 200, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250,1300, 1350, 1400, 1500 nucleotides, or up to the number of nucleotidespresent in a full-length cytidine deaminase-like nucleotide sequencedisclosed herein (for example, 1585 nucleotides for SEQ ID NO:1 or 3)depending upon the intended use.

[0091] Alternatively, a nucleic acid molecules that is a fragment of ancytidine deaminase-like nucleotide sequence of the present inventioncomprises a nucleotide sequence consisting of nucleotides 1-100,100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900,900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1585 of SEQID NO:1 or 3.

[0092] It is understood that isolated fragments include any contiguoussequence not disclosed prior to the invention as well as sequences thatare substantially the same and which are not disclosed. Accordingly, ifan isolated fragment is disclosed prior to the present invention, thatfragment is not intended to be encompassed by the invention. When asequence is not disclosed prior to the present invention, an isolatednucleic acid fragment is at least about 12, 15, 20, 25, or 30 contiguousnucleotides. Other regions of the nucleotide sequence may comprisefragments of various sizes, depending upon potential homology withpreviously disclosed sequences.

[0093] A fragment of an cytidine deaminase-like nucleotide sequence thatencodes a biologically active portion of an cytidine deaminase-likeprotein of the invention will encode at least about 15, 25, 30, 50, 75,100, 125, 150, 175, 200, 250, or 300 contiguous amino acids, or up tothe total number of amino acids present in a full-length cytidinedeaminase-like protein of the invention (for example, 339 amino acidsfor SEQ ID NO:2). Fragments of an cytidine deaminase-like nucleotidesequence that are useful as hybridization probes for PCR primersgenerally need not encode a biologically active portion of an cytidinedeaminase-like protein.

[0094] Alternatively, a fragment of a polypeptide of the presentinvention comprises an amino acid sequence consisting of amino acidresidues 1-20, 20-40, 40-60, 60-80, 80-100, 100-120, 120-140, 140-160,160-180, 180-200, 200-220, 220-240, 240-260, 260-280, 280-300, 300-320,320-340 of SEQ ID NO:2.

[0095] Nucleic acid molecules that are variants of the cytidinedeaminase-like nucleotide sequences disclosed herein are alsoencompassed by the present invention. “Variants” of the cytidinedeaminase-like nucleotide sequences include those sequences that encodethe cytidine deaminase-like proteins disclosed herein but that differconservatively because of the degeneracy of the genetic code. Thesenaturally occurring allelic variants can be identified with the use ofwell-known molecular biology techniques, such as polymerase chainreaction (PCR) and hybridization techniques as outlined below. Variantnucleotide sequences also include synthetically derived nucleotidesequences that have been generated, for example, by using site-directedmutagenesis but which still encode the cytidine deaminase-like proteinsdisclosed in the present invention as discussed below. Generally,nucleotide sequence variants of the invention will have at least about45%, 55%, 65%, 75%, 85%, 95%, or 98% identity to a particular nucleotidesequence disclosed herein. A variant cytidine deaminase-like nucleotidesequence will encode an cytidine deaminase-like protein that has anamino acid sequence having at least about 45%, 55%, 65%, 75%, 85%, 95%,or 98% identity to the amino acid sequence of an cytidine deaminase-likeprotein disclosed herein.

[0096] In addition to the cytidine deaminase-like nucleotide sequencesshown in SEQ ID NOs:1 and 3, it will be appreciated by those skilled inthe art that DNA sequence polymorphisms that lead to changes in theamino acid sequences of cytidine deaminase-like proteins may existwithin a population (e.g., the human population). Such geneticpolymorphism in an cytidine deaminase-like gene may exist amongindividuals within a population due to natural allelic variation. Anallele is one of a group of genes that occur alternatively at a givengenetic locus. As used herein, the terms “gene” and “recombinant gene”refer to nucleic acid molecules comprising an open reading frameencoding an cytidine deaminase-like protein, preferably a mammaliancytidine deaminase-like protein. As used herein, the phrase “allelicvariant” refers to a nucleotide sequence that occurs at an cytidinedeaminase-like locus or to a polypeptide encoded by the nucleotidesequence. Such natural allelic variations can typically result in 1-5%variance in the nucleotide sequence of the cytidine deaminase-like gene.Any and all such nucleotide variations and resulting amino acidpolymorphisms or variations in an cytidine deaminase-like sequence thatare the result of natural allelic variation and that do not alter thefunctional activity of cytidine deaminase-like proteins are intended tobe within the scope of the invention.

[0097] Moreover, nucleic acid molecules encoding cytidine deaminase-likeproteins from other species (cytidine deaminase-like homologues), whichhave a nucleotide sequence differing from that of the cytidinedeaminase-like sequences disclosed herein, are intended to be within thescope of the invention. For example, nucleic acid moleculescorresponding to natural allelic variants and homologues of the humancytidine deaminase-like cDNA of the invention can be isolated based ontheir identity to the human cytidine deaminase-like nucleic aciddisclosed herein using the human cDNA, or a portion thereof, as ahybridization probe according to standard hybridization techniques understringent hybridization conditions as disclosed below.

[0098] In addition to naturally-occurring allelic variants of thecytidine deaminase-like sequences that may exist in the population, theskilled artisan will further appreciate that changes can be introducedby mutation into the nucleotide sequences of the invention therebyleading to changes in the amino acid sequence of the encoded cytidinedeaminase-like proteins, without altering the biological activity of thecytidine deaminase-like proteins. Thus, an isolated nucleic acidmolecule encoding an cytidine deaminase-like protein having a sequencethat differs from that of SEQ ID NO:2 can be created by introducing oneor more nucleotide substitutions, additions, or deletions into thecorresponding nucleotide sequence disclosed herein, such that one ormore amino acid substitutions, additions or deletions are introducedinto the encoded protein. Mutations can be introduced by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Such variant nucleotide sequences are also encompassed bythe present invention.

[0099] For example, preferably, conservative amino acid substitutionsmay be made at one or more predicted, preferably nonessential amino acidresidues. A “nonessential” amino acid residue is a residue that can bealtered from the wild-type sequence of an cytidine deaminase-likeprotein (e.g., the sequence of SEQ ID NO:2) without altering thebiological activity, whereas an “essential” amino acid residue isrequired for biological activity. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

[0100] Alternatively, variant cytidine deaminase-like nucleotidesequences can be made by introducing mutations randomly along all orpart of an cytidine deaminase-like coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forcytidine deaminase-like biological activity to identify mutants thatretain activity. Following mutagenesis, the encoded protein can beexpressed recombinantly, and the activity of the protein can bedetermined using standard assay techniques.

[0101] Thus the nucleotide sequences of the invention include thesequences disclosed herein as well as fragments and variants thereof.The cytidine deaminase-like nucleotide sequences of the invention, andfragments and variants thereof, can be used as probes and/or primers toidentify and/or clone cytidine deaminase-like homologues in other celltypes, e.g., from other tissues, as well as cytidine deaminase-likehomologues from other mammals. Such probes can be used to detecttranscripts or genomic sequences encoding the same or identicalproteins. These probes can be used as part of a diagnostic test kit foridentifying cells or tissues that misexpress an cytidine deaminase-likeprotein, such as by measuring levels of an cytidinedeaminase-like-encoding nucleic acid in a sample of cells from asubject, e.g., detecting cytidine deaminase-like mRNA levels ordetermining whether a genomic cytidine deaminase-like gene has beenmutated or deleted.

[0102] In this manner, methods such as PCR, hybridization, and the likecan be used to identify such sequences having substantial identity tothe sequences of the invention. See, for example, Sambrook et al. (1989)Molecular Cloning: Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.) and Innis, et al. (1990) PCRProtocols: A Guide to Methods and Applications (Academic Press, NY).cytidine deaminase-like nucleotide sequences isolated based on theirsequence identity to the cytidine deaminase-like nucleotide sequencesset forth herein or to fragments and variants thereof are encompassed bythe present invention.

[0103] In a hybridization method, all or part of a known cytidinedeaminase-like nucleotide sequence can be used to screen cDNA or genomiclibraries. Methods for construction of such cDNA and genomic librariesare generally known in the art and are disclosed in Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold SpringHarbor Laboratory Press, Plainview, N.Y.). The so-called hybridizationprobes may be genomic DNA fragments, cDNA fragments, RNA fragments, orother oligonucleotides, and may be labeled with a detectable group suchas ³²P, or any other detectable marker, such as other radioisotopes, afluorescent compound, an enzyme, or an enzyme co-factor. Probes forhybridization can be made by labeling synthetic oligonucleotides basedon the known cytidine deaminase-like nucleotide sequence disclosedherein. Degenerate primers designed on the basis of conservednucleotides or amino acid residues in a known cytidine deaminase-likenucleotide sequence or encoded amino acid sequence can additionally beused. The probe typically comprises a region of nucleotide sequence thathybridizes under stringent conditions to at least about 12, preferablyabout 25, more preferably about 50, 75, 100, 125, 150, 175, 200, 250,300, 350, or 400 consecutive nucleotides of an cytidine deaminase-likenucleotide sequence of the invention or a fragment or variant thereof.Preparation of probes for hybridization is generally known in the artand is disclosed in Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.), herein incorporated by reference.

[0104] For example, in one embodiment, a previously unidentifiedcytidine deaminase-like nucleic acid molecule hybridizes under stringentconditions to a probe that is a nucleic acid molecule comprising one ofthe cytidine deaminase-like nucleotide sequences of the invention or afragment thereof. In another embodiment, the previously unknown cytidinedeaminase-like nucleic acid molecule is at least about 300, 325, 350,375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 2,000,3,000, 4,000 or 5,000 nucleotides in length and hybridizes understringent conditions to a probe that is a nucleic acid moleculecomprising one of the cytidine deaminase-like nucleotide sequencesdisclosed herein or a fragment thereof.

[0105] Accordingly, in another embodiment, an isolated previouslyunknown cytidine deaminase-like nucleic acid molecule of the inventionis at least about 300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650,700, 800, 900, 1000, 1,100, 1,200, 1,300, or 1,400 nucleotides in lengthand hybridizes under stringent conditions to a probe that is a nucleicacid molecule comprising one of the nucleotide sequences of theinvention, preferably the coding sequence set forth in SEQ ID NO:1 or 3or a complement, fragment, or variant thereof.

[0106] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences typically remain hybridized to each other.Such stringent conditions are known to those skilled in the art and canbe found in Current Protocols in Molecular Biology (John Wiley & Sons,New York (1989)), 6.3.1-6.3.6. A preferred, example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50° C. Another example of stringent hybridizationconditions are hybridization in 6× sodium chloride/sodium citrate (SSC)at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at55° C. A further example of stringent hybridization conditions arehybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.Preferably, stringent hybridization conditions are hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 65° C. Particularly preferredstringency conditions (and the conditions that should be used if thepractitioner is uncertain about what conditions should be applied todetermine if a molecule is within a hybridization limitation of theinvention) are 0.5M Sodium Phosphate, 7% SDS at 65° C., followed by oneor more washes at 0.2×SSC, 1% SDS at 65° C. Preferably, an isolatednucleic acid molecule that hybridizes under stringent conditions to ancytidine deaminase-like sequence of the invention corresponds to anaturally-occurring nucleic acid molecule. As used herein, a“naturally-occurring” nucleic acid molecule refers to an RNA or DNAmolecule having a nucleotide sequence that occurs in nature (e.g.,encodes a natural protein).

[0107] Thus, in addition to the cytidine deaminase-like nucleotidesequences disclosed herein and fragments and variants thereof, theisolated nucleic acid molecules of the invention also encompasshomologous DNA sequences identified and isolated from other cells and/ororganisms by hybridization with entire or partial sequences obtainedfrom the cytidine deaminase-like nucleotide sequences disclosed hereinor variants and fragments thereof.

[0108] The present invention also encompasses antisense nucleic acidmolecules, i.e., molecules that are complementary to a sense nucleicacid encoding a protein, e.g., complementary to the coding strand of adouble-stranded cDNA molecule, or complementary to an mRNA sequence.Accordingly, an antisense nucleic acid can hydrogen bond to a sensenucleic acid. The antisense nucleic acid can be complementary to anentire cytidine deaminase-like coding strand, or to only a portionthereof, e.g., all or part of the protein coding region (or open readingframe). An antisense nucleic acid molecule can be antisense to anoncoding region of the coding strand of a nucleotide sequence encodingan cytidine deaminase-like protein. The noncoding regions are the 5′ and3′ sequences that flank the coding region and are not translated intoamino acids.

[0109] Given the coding-strand sequence encoding an cytidinedeaminase-like protein disclosed herein (e.g., SEQ ID NO:1 or 3),antisense nucleic acids of the invention can be designed according tothe rules of Watson and Crick base pairing. The antisense nucleic acidmolecule can be complementary to the entire coding region of cytidinedeaminase-like mRNA, but more preferably is an oligonucleotide that isantisense to only a portion of the coding or noncoding region ofcytidine deaminase-like mRNA. For example, the antisense oligonucleotidecan be complementary to the region surrounding the translation startsite of cytidine deaminase-like mRNA. An antisense oligonucleotide canbe, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50nucleotides in length. An antisense nucleic acid of the invention can beconstructed using chemical synthesis and enzymatic ligation proceduresknown in the art.

[0110] For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, including, but not limited to, for example e.g., phosphorothioatederivatives and acridine substituted nucleotides. Alternatively, theantisense nucleic acid can be produced biologically using an expressionvector into which a nucleic acid has been subcloned in an antisenseorientation (i.e., RNA transcribed from the inserted nucleic acid willbe of an antisense orientation to a target nucleic acid of interest,described further in the following subsection).

[0111] When used therapeutically, the antisense nucleic acid moleculesof the invention are typically administered to a subject or generated insitu such that they hybridize with or bind to cellular mRNA and/orgenomic DNA encoding an cytidine deaminase-like protein to therebyinhibit expression of the protein, e.g., by inhibiting transcriptionand/or translation. An example of a route of administration of antisensenucleic acid molecules of the invention includes direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, antisense molecules can be linked to peptides or antibodiesto form a complex that specifically binds to receptors or antigensexpressed on a selected cell surface. The antisense nucleic acidmolecules can also be delivered to cells using the vectors describedherein. To achieve sufficient intracellular concentrations of theantisense molecules, vector constructs in which the antisense nucleicacid molecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

[0112] An antisense nucleic acid molecule of the invention can be anα-anomeric nucleic acid molecule. An α-anomeric nucleic acid moleculeforms specific double-stranded hybrids with complementary RNA in which,contrary to the usual β-units, the strands run parallel to each other(Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

[0113] The invention also encompasses ribozymes, which are catalytic RNAmolecules with ribonuclease activity that are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Ribozymes (e.g., hammerhead ribozymes (describedin Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used tocatalytically cleave cytidine deaminase-like mRNA transcripts to therebyinhibit translation of cytidine deaminase-like mRNA. A ribozyme havingspecificity for an cytidine deaminase-like-encoding nucleic acid can bedesigned based upon the nucleotide sequence of an cytidinedeaminase-like cDNA disclosed herein (e.g., SEQ ID NO:1 or 3). See,e.g., Cech et al., U.S. Pat. No. 4,987,071; and Cech et al., U.S. Pat.No. 5,116,742. Alternatively, cytidine deaminase-like mRNA can be usedto select a catalytic RNA having a specific ribonuclease activity from apool of RNA molecules. See, e.g., Bartel and Szostak (1993) Science261:1411-1418.

[0114] The invention also encompasses nucleic acid molecules that formtriple helical structures. For example, cytidine deaminase-like geneexpression can be inhibited by targeting nucleotide sequencescomplementary to the regulatory region of the cytidine deaminase-likeprotein (e.g., the cytidine deaminase-like promoter and/or enhancers) toform triple helical structures that prevent transcription of thecytidine deaminase-like gene in target cells. See generally Helene(1991) Anticancer Drug Des. 6(6):569; Helene (1992) Ann. N.Y. Acad. Sci.660:27; and Maher (1992) Bioassays 14(12):807.

[0115] In preferred embodiments, the nucleic acid molecules of theinvention can be modified at the base moiety, sugar moiety, or phosphatebackbone to improve, e.g., the stability, hybridization, or solubilityof the molecule. For example, the deoxyribose phosphate backbone of thenucleic acids can be modified to generate peptide nucleic acids (seeHyrup et al. (1996) Bioorganic & Medicinal Chemistry 4:5). As usedherein, the terms “peptide nucleic acids” or “PNAs” refer to nucleicacid mimics, e.g., DNA mimics, in which the deoxyribose phosphatebackbone is replaced by a pseudopeptide backbone and only the fournatural nucleobases are retained. The neutral backbone of PNAs has beenshown to allow for specific hybridization to DNA and RNA underconditions of low ionic strength. The synthesis of PNA oligomers can beperformed using standard solid-phase peptide synthesis protocols asdescribed, for example, in Hyrup et al. (1996), supra; Perry-O'Keefe etal. (1996) Proc. Natl. Acad. Sci. USA 93:14670.

[0116] PNAs of an cytidine deaminase-like molecule can be used intherapeutic and diagnostic applications. For example, PNAs can be usedas antisense or antigene agents for sequence-specific modulation of geneexpression by, e.g., inducing transcription or translation arrest orinhibiting replication. PNAs of the invention can also be used, e.g., inthe analysis of single base pair mutations in a gene by, e.g.,PNA-directed PCR clamping; as artificial restriction enzymes when usedin combination with other enzymes, e.g., S1 nucleases (Hyrup (1996),supra); or as probes or primers for DNA sequence and hybridization(Hyrup (1996), supra; Perry-O'Keefe et al. (1996), supra).

[0117] In another embodiment, PNAs of an cytidine deaminase-likemolecule can be modified, e.g., to enhance their stability, specificity,or cellular uptake, by attaching lipophilic or other helper groups toPNA, by the formation of PNA-DNA chimeras, or by the use of liposomes orother techniques of drug delivery known in the art. The synthesis ofPNA-DNA chimeras can be performed as described in Hyrup (1996), supra;Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63; Mag et al. (1989)Nucleic Acids Res. 17:5973; and Peterson et al. (1975) Bioorganic Med.Chem. Lett. 5:1119.

[0118] II. Isolated Cytidine Deaminase-Like Proteins and Anti-CytidineDeaminase-Like Antibodies

[0119] Cytidine deaminase-like proteins are also encompassed within thepresent invention. By “cytidine deaminase-like protein” is intended aprotein having the amino acid sequence set forth in SEQ ID NO: 2, aswell as fragments, biologically active portions, and variants thereof.

[0120] “Fragments” or “biologically active portions” include polypeptidefragments suitable for use as immunogens to raise anti-cytidinedeaminase-like antibodies. Fragments include peptides comprising aminoacid sequences sufficiently identical to or derived from the amino acidsequence of an cytidine deaminase-like protein, or partial-lengthprotein, of the invention and exhibiting at least one activity of ancytidine deaminase-like protein, but which include fewer amino acidsthan the full-length (SEQ ID NO:2) cytidine deaminase-like proteindisclosed herein. Typically, biologically active portions comprise adomain or motif with at least one activity of the cytidinedeaminase-like protein. A biologically active portion of an cytidinedeaminase-like protein can be a polypeptide which is, for example, 10,25, 50, 100 or more amino acids in length. Such biologically activeportions can be prepared by recombinant techniques and evaluated for oneor more of the functional activities of a native cytidine deaminase-likeprotein. As used here, a fragment comprises at least 5 contiguous aminoacids of SEQ ID NO:2. The invention encompasses other fragments,however, such as any fragment in the protein greater than 6, 7, 8, or 9amino acids.

[0121] By “variants” is intended proteins or polypeptides having anamino acid sequence that is at least about 45%, 55%, 65%, preferablyabout 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQID NO:3. Variants also include polypeptides encoded by a nucleic acidmolecule that hybridizes to the nucleic acid molecule of SEQ ID NO:1 or3, or a complement thereof, under stringent conditions. In anotherembodiment, a variant of an isolated polypeptide of the presentinvention differs, by at least 1, but less than 5, 10, 20, 50, or 100amino acid residues from the sequence shown in SEQ ID NO:2. If alignmentis needed for this comparison the sequences should be aligned formaximum identity. “Looped” out sequences from deletions or insertions,or mismatches, are considered differences. Such variants generallyretain the functional activity of the cytidine deaminase-like proteinsof the invention. Variants include polypeptides that differ in aminoacid sequence due to natural allelic variation or mutagenesis.

[0122] The invention also provides cytidine deaminase-like chimeric orfusion proteins. As used herein, an cytidine deaminase-like “chimericprotein” or “fusion protein” comprises an cytidine deaminase-likepolypeptide operably linked to a non-cytidine deaminase-likepolypeptide. A “cytidine deaminase-like polypeptide” refers to apolypeptide having an amino acid sequence corresponding to an cytidinedeaminase-like protein, whereas a “non-cytidine deaminase-likepolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to a protein that is not substantially identical to thecytidine deaminase-like protein, e.g., a protein that is different fromthe cytidine deaminase-like protein and which is derived from the sameor a different organism. Within an cytidine deaminase-like fusionprotein, the cytidine deaminase-like polypeptide can correspond to allor a portion of an cytidine deaminase-like protein, preferably at leastone biologically active portion of an cytidine deaminase-like protein.Within the fusion protein, the term “operably linked” is intended toindicate that the cytidine deaminase-like polypeptide and thenon-cytidine deaminase-like polypeptide are fused in-frame to eachother. The non-cytidine deaminase-like polypeptide can be fused to theN-terminus or C-terminus of the cytidine deaminase-like polypeptide.

[0123] One useful fusion protein is a GST-cytidine deaminase-like fusionprotein in which the cytidine deaminase-like sequences are fused to theC-terminus of the GST sequences. Such fusion proteins can facilitate thepurification of recombinant cytidine deaminase-like proteins.

[0124] In yet another embodiment, the fusion protein is an cytidinedeaminase-like-immunoglobulin fusion protein in which all or part of ancytidine deaminase-like protein is fused to sequences derived from amember of the immunoglobulin protein family. The cytidinedeaminase-like-immunoglobulin fusion proteins of the invention can beincorporated into pharmaceutical compositions and administered to asubject to inhibit an interaction between an cytidine deaminase-likeligand and an cytidine deaminase-like protein on the surface of a cell,thereby suppressing cytidine deaminase-like-mediated signal transductionin vivo. The cytidine deaminase-like-immunoglobulin fusion proteins canbe used to affect the bioavailability of an cytidine deaminase-likecognate ligand. Inhibition of the cytidine deaminase-likeligand/cytidine deaminase-like interaction may be usefultherapeutically. Moreover, the cytidine deaminase-like-immunoglobulinfusion proteins of the invention can be used as immunogens to produceanti-cytidine deaminase-like antibodies in a subject, to purify cytidinedeaminase-like ligands, and in screening assays to identify moleculesthat inhibit the interaction of an cytidine deaminase-like protein withan cytidine deaminase-like ligand.

[0125] Preferably, an cytidine deaminase-like chimeric or fusion proteinof the invention is produced by standard recombinant DNA techniques. Forexample, DNA fragments coding for the different polypeptide sequencesmay be ligated together in-frame, or the fusion gene can be synthesized,such as with automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primersthat give rise to complementary overhangs between two consecutive genefragments, which can subsequently be annealed and reamplified togenerate a chimeric gene sequence (see, e.g., Ausubel et al., eds.(1995) Current Protocols in Molecular Biology) (Greene Publishing andWiley-Interscience, NY). Moreover, an cytidine deaminase-like-encodingnucleic acid can be cloned into a commercially available expressionvector such that it is linked in-frame to an existing fusion moiety.

[0126] Variants of the cytidine deaminase-like proteins can function aseither cytidine deaminase-like agonists (mimetics) or as cytidinedeaminase-like antagonists. Variants of the cytidine deaminase-likeprotein can be generated by mutagenesis, e.g., discrete point mutationor truncation of the cytidine deaminase-like protein. An agonist of thecytidine deaminase-like protein can retain substantially the same, or asubset, of the biological activities of the naturally occurring form ofthe cytidine deaminase-like protein. An antagonist of the cytidinedeaminase-like protein can inhibit one or more of the activities of thenaturally occurring form of the cytidine deaminase-like protein by, forexample, competitively binding to a downstream or upstream member of acellular signaling cascade that includes the cytidine deaminase-likeprotein. Thus, specific biological effects can be elicited by treatmentwith a variant of limited function. Treatment of a subject with avariant having a subset of the biological activities of the naturallyoccurring form of the protein can have fewer side effects in a subjectrelative to treatment with the naturally occurring form of the cytidinedeaminase-like proteins.

[0127] Variants of an cytidine deaminase-like protein that function aseither cytidine deaminase-like agonists or as cytidine deaminase-likeantagonists can be identified by screening combinatorial libraries ofmutants, e.g., truncation mutants, of an cytidine deaminase-like proteinfor cytidine deaminase-like protein agonist or antagonist activity. Inone embodiment, a variegated library of cytidine deaminase-like variantsis generated by combinatorial mutagenesis at the nucleic acid level andis encoded by a variegated gene library. A variegated library ofcytidine deaminase-like variants can be produced by, for example,enzymatically ligating a mixture of synthetic oligonucleotides into genesequences such that a degenerate set of potential cytidinedeaminase-like sequences is expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay) containing the set of cytidine deaminase-like sequencestherein. There are a variety of methods that can be used to producelibraries of potential cytidine deaminase-like variants from adegenerate oligonucleotide sequence. Chemical synthesis of a degenerategene sequence can be performed in an automatic DNA synthesizer, and thesynthetic gene then ligated into an appropriate expression vector. Useof a degenerate set of genes allows for the provision, in one mixture,of all of the sequences encoding the desired set of potential cytidinedeaminase-like sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang (1983)Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic AcidRes. 11:477).

[0128] In addition, libraries of fragments of an cytidine deaminase-likeprotein coding sequence can be used to generate a variegated populationof cytidine deaminase-like fragments for screening and subsequentselection of variants of an cytidine deaminase-like protein. In oneembodiment, a library of coding sequence fragments can be generated bytreating a double-stranded PCR fragment of an cytidine deaminase-likecoding sequence with a nuclease under conditions wherein nicking occursonly about once per molecule, denaturing the double-stranded DNA,renaturing the DNA to form double-stranded DNA which can includesense/antisense pairs from different nicked products, removingsingle-stranded portions from reformed duplexes by treatment with S1nuclease, and ligating the resulting fragment library into an expressionvector. By this method, one can derive an expression library thatencodes N-terminal and internal fragments of various sizes of thecytidine deaminase-like protein.

[0129] Several techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations ortruncation and for screening cDNA libraries for gene products having aselected property. Such techniques are adaptable for rapid screening ofthe gene libraries generated by the combinatorial mutagenesis ofcytidine deaminase-like proteins. The most widely used techniques, whichare amenable to high through-put analysis, for screening large genelibraries typically include cloning the gene library into replicableexpression vectors, transforming appropriate cells with the resultinglibrary of vectors, and expressing the combinatorial genes underconditions in which detection of a desired activity facilitatesisolation of the vector encoding the gene whose product was detected.Recursive ensemble mutagenesis (REM), a technique that enhances thefrequency of functional mutants in the libraries, can be used incombination with the screening assays to identify cytidinedeaminase-like variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci.USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering6(3):327-331).

[0130] An isolated cytidine deaminase-like polypeptide of the inventioncan be used as an immunogen to generate antibodies that bind cytidinedeaminase-like proteins using standard techniques for polyclonal andmonoclonal antibody preparation. The full-length cytidine deaminase-likeprotein can be used or, alternatively, the invention provides antigenicpeptide fragments of cytidine deaminase-like proteins for use asimmunogens. The antigenic peptide of an cytidine deaminase-like proteincomprises at least 8, preferably 10, 15, 20, or 30 amino acid residuesof the amino acid sequence shown in SEQ ID NO:2 and encompasses anepitope of an cytidine deaminase-like protein such that an antibodyraised against the peptide forms a specific immune complex with thecytidine deaminase-like protein. Preferred epitopes encompassed by theantigenic peptide are regions of a cytidine deaminase-like protein thatare located on the surface of the protein, e.g., hydrophilic regions.

[0131] Accordingly, another aspect of the invention pertains toanti-cytidine deaminase-like polyclonal and monoclonal antibodies thatbind an cytidine deaminase-like protein. Polyclonal anti-cytidinedeaminase-like antibodies can be prepared by immunizing a suitablesubject (e.g., rabbit, goat, mouse, or other mammal) with an cytidinedeaminase-like immunogen. The anti-cytidine deaminase-like antibodytiter in the immunized subject can be monitored over time by standardtechniques, such as with an enzyme linked immunosorbent assay (ELISA)using immobilized cytidine deaminase-like protein. At an appropriatetime after immunization, e.g., when the anti-cytidine deaminase-likeantibody titers are highest, antibody-producing cells can be obtainedfrom the subject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (1975) Nature 256:495-497, the human B cellhybridoma technique (Kozbor et al. (1983) Immunol. Today 4:72), theEBV-hybridoma technique (Cole et al. (1985) in Monoclonal Antibodies andCancer Therapy, ed. Reisfeld and Sell (Alan R. Liss, Inc., New York,N.Y.), pp. 77-96) or trioma techniques. The technology for producinghybridomas is well known (see generally Coligan et al., eds. (1994)Current Protocols in Immunology (John Wiley & Sons, Inc., New York,N.Y.); Galfre et al. (1977) Nature 266:55052; Kenneth (1980) inMonoclonal Antibodies: A New Dimension In Biological Analyses (PlenumPublishing Corp., NY; and Lerner (1981) Yale J. Biol. Med., 54:387-402).

[0132] Alternative to preparing monoclonal antibody-secretinghybridomas, a monoclonal anti-cytidine deaminase-like antibody can beidentified and isolated by screening a recombinant combinatorialimmunoglobulin library (e.g., an antibody phage display library) with ancytidine deaminase-like protein to thereby isolate immunoglobulinlibrary members that bind the cytidine deaminase-like protein. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit,Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening antibodydisplay library can be found in, for example, U.S. Pat. No. 5,223,409;PCT Publication Nos. WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679;93/01288; WO 92/01047; 92/09690; and 90/02809; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J. 12:725-734.

[0133] Additionally, recombinant anti-cytidine deaminase-likeantibodies, such as chimeric and humanized monoclonal antibodies,comprising both human and nonhuman portions, which can be made usingstandard recombinant DNA techniques, are within the scope of theinvention. Such chimeric and humanized monoclonal antibodies can beproduced by recombinant DNA techniques known in the art, for exampleusing methods described in PCT Publication Nos. WO 86/101533 and WO87/02671; European Patent Application Nos. 184,187, 171,496, 125,023,and 173,494; U.S. Pat. Nos. 4,816,567 and 5,225,539; European PatentApplication 125,023; Better et al. (1988) Science 240:1041-1043; Liu etal. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986)Bio/Techniques 4:214; Jones et al. (1986) Nature 321:552-525; Verhoeyanet al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.141:4053-4060.

[0134] Completely human antibodies are particularly desirable fortherapeutic treatment of human patients. Such antibodies can be producedusing transgenic mice that are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheavy and light chain genes. See, for example, Lonberg and Huszar (1995)Int. Rev. Immunol. 13:65-93); and U.S. Pat. Nos. 5,625,126; 5,633,425;5,569,825; 5,661,016; and 5,545,806. In addition, companies such asAbgenix, Inc. (Fremont, Calif.), can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above.

[0135] Completely human antibodies that recognize a selected epitope canbe generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. This technology is described by Jespers etal. (1994) Bio/Technology 12:899-903).

[0136] An anti-cytidine deaminase like antibody (e.g., monoclonalantibody) can be used to isolate cytidine deaminase-like proteins bystandard techniques, such as affinity chromatography orimmunoprecipitation. An anti-cytidine deaminase-like antibody canfacilitate the purification of natural cytidine deaminase-like proteinfrom cells and of recombinantly produced cytidine deaminase-like proteinexpressed in host cells. Moreover, an anti-cytidine deaminase-likeantibody can be used to detect cytidine deaminase-like protein (e.g., ina cellular lysate or cell supernatant) in order to evaluate theabundance and pattern of expression of the cytidine deaminase-likeprotein. Anti-cytidine deaminase-like antibodies can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling theantibody to a detectable substance. Examples of detectable substancesinclude various enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S,or ³H.

[0137] Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). The conjugates of the invention canbe used for modifying a given biological response, the drug moiety isnot to be construed as limited to classical chemical therapeutic agents.For example, the drug moiety may be a protein or polypeptide possessinga desired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, alpha-interferon,beta-interferon, nerve growth factor, platelet derived growth factor,tissue plasminogen activator; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

[0138] Techniques for conjugating such therapeutic moiety to antibodiesare well known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies'84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can beconjugated to a second antibody to form an antibody heteroconjugate asdescribed by Segal in U.S. Pat. No. 4,676,980.

[0139] III. Recombinant Expression Vectors and Host Cells

[0140] Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding an cytidinedeaminase-like protein (or a portion thereof). “Vector” refers to anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked, such as a “plasmid”, a circulardouble-stranded DNA loop into which additional DNA segments can beligated, or a viral vector, where additional DNA segments can be ligatedinto the viral genome. The vectors are useful for autonomous replicationin a host cell or may be integrated into the genome of a host cell uponintroduction into the host cell, and thereby are replicated along withthe host genome (e.g., nonepisomal mammalian vectors). Expressionvectors are capable of directing the expression of genes to which theyare operably linked. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of plasmids (vectors).However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses, and adeno-associated viruses), that serveequivalent functions.

[0141] The recombinant expression vectors of the invention comprise anucleic acid of the invention in a form suitable for expression of thenucleic acid in a host cell. This means that the recombinant expressionvectors include one or more regulatory sequences, selected on the basisof the host cells to be used for expression, operably linked to thenucleic acid sequence to be expressed. “Operably linked” is intended tomean that the nucleotide sequence of interest is linked to theregulatory sequence(s) in a manner that allows for expression of thenucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell). The term “regulatory sequence” is intended to include promoters,enhancers, and other expression control elements (e.g., polyadenylationsignals). See, for example, Goeddel (1990) in Gene ExpressionTechnology: Methods in Enzymology 185 (Academic Press, San Diego,Calif.). Regulatory sequences include those that direct constitutiveexpression of a nucleotide sequence in many types of host cell and thosethat direct expression of the nucleotide sequence only in certain hostcells (e.g., tissue-specific regulatory sequences). It will beappreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, etc.The expression vectors of the invention can be introduced into hostcells to thereby produce proteins or peptides, including fusion proteinsor peptides, encoded by nucleic acids as described herein (e.g.,cytidine deaminase-like proteins, mutant forms of cytidinedeaminase-like proteins, fusion proteins, etc.).

[0142] It is further recognized that the nucleic acid sequences of theinvention can be altered to contain codons, which are preferred, or nonpreferred, for a particular expression system. For example, the nucleicacid can be one in which at least one altered codon, and preferably atleast 10%, or 20% of the codons have been altered such that the sequenceis optimized for expression in E. coli, yeast, human, insect, or CHOcells. Methods for determining such codon usage are well known in theart.

[0143] The recombinant expression vectors of the invention can bedesigned for expression of cytidine deaminase-like protein inprokaryotic or eukaryotic host cells. Expression of proteins inprokaryotes is most often carried out in E. coli with vectors containingconstitutive or inducible promoters directing the expression of eitherfusion or nonfusion proteins. Fusion vectors add a number of amino acidsto a protein encoded therein, usually to the amino terminus of therecombinant protein. Typical fusion expression vectors include pGEX(Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67:31-40), pMAL(New England Biolabs, Beverly, Mass.), and pRIT5 (Pharmacia, Piscataway,N.J.) which fuse glutathione S-transferase (GST), maltose E bindingprotein, or protein A, respectively, to the target recombinant protein.Examples of suitable inducible nonfusion E. coli expression vectorsinclude pTrc (Amann et al. (1988) Gene 69:301-315) and pET 11d (Studieret al. (1990) in Gene Expression Technology: Methods in Enzymology 185(Academic Press, San Diego, Calif.), pp. 60-89). Strategies to maximizerecombinant protein expression in E. coli can be found in Gottesman(1990) in Gene Expression Technology: Methods in Enzymology 185(Academic Press, CA), pp. 119-128 and Wada et al. (1992) Nucleic AcidsRes. 20:2111-2118. Target gene expression from the pTrc vector relies onhost RNA polymerase transcription from a hybrid trp-lac fusion promoter.

[0144] Suitable eukaryotic host cells include insect cells (examples ofBaculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf 9 cells) include the pAc series (Smith et al.(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow andSummers (1989) Virology 170:31-39)); yeast cells (examples of vectorsfor expression in yeast S. cereivisiae include pYepSec1 (Baldari et al.(1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2(Invitrogen Corporation, San Diego, Calif.), and pPicZ (InvitrogenCorporation, San Diego, Calif.)); or mammalian cells (mammalianexpression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC(Kaufman et al. (1987) EMBO J. 6:187:195)). Suitable mammalian cellsinclude Chinese hamster ovary cells (CHO) or COS cells. In mammaliancells, the expression vector's control functions are often provided byviral regulatory elements. For example, commonly used promoters arederived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus40. For other suitable expression systems for both prokaryotic andeukaryotic cells, see chapters 16 and 17 of Sambrook et al. (1989)Molecular cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.). See, Goeddel (1990) in GeneExpression Technology: Methods in Enzymology 185 (Academic Press, SanDiego, Calif.). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

[0145] The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell but are stillincluded within the scope of the term as used herein.

[0146] A “purified preparation of cells”, as used herein, refers to, inthe case of plant or animal cells, an in vitro preparation of cells andnot an entire intact plant or animal. In the case of cultured cells ormicrobial cells, it consists of a preparation of at least 10% and morepreferably 50% of the subject cells.

[0147] In one embodiment, the expression vector is a recombinantmammalian expression vector that comprises tissue-specific regulatoryelements that direct expression of the nucleic acid preferentially in aparticular cell type. Suitable tissue-specific promoters include thealbumin promoter (e.g., liver-specific promoter; Pinkert et al. (1987)Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton(1988) Adv. Immunol. 43:235-275), in particular promoters of T cellreceptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European Application PatentPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox homeobox promoters (Kessel andGruss (1990) Science 249:374-379), the α-fetoprotein promoter (Campesand Tilghman (1989) Genes Dev. 3:537-546), and the like.

[0148] The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperably linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to cytidine deaminase-like mRNA. Regulatory sequencesoperably linked to a nucleic acid cloned in the antisense orientationcan be chosen to direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen to direct constitutive,tissue-specific, or cell-type-specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid, or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see Weintraub et al. (1986)Reviews-Trends in Genetics, Vol. 1(1).

[0149] Vector DNA can be introduced into prokaryotic or eukaryotic cellsvia conventional transformation or transfection techniques. As usedherein, the terms “transformation” and “transfection” are intended torefer to a variety of art-recognized techniques for introducing foreignnucleic acid (e.g., DNA) into a host cell, including calcium phosphateor calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, or electroporation. Suitable methods fortransforming or transfecting host cells can be found in Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold SpringHarbor Laboratory Press, Plainview, N.Y.) and other laboratory manuals.

[0150] For stable transfection of mammalian cells, it is known that,depending upon the expression vector and transfection technique used,only a small fraction of cells may integrate the foreign DNA into theirgenome. In order to identify and select these integrants, a gene thatencodes a selectable marker (e.g., for resistance to antibiotics) isgenerally introduced into the host cells along with the gene ofinterest. Preferred selectable markers include those which conferresistance to drugs, such as G418, hygromycin, and methotrexate. Nucleicacid encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding an cytidine deaminase-like protein orcan be introduced on a separate vector. Cells stably transfected withthe introduced nucleic acid can be identified by drug selection (e.g.,cells that have incorporated the selectable marker gene will survive,while the other cells die).

[0151] A host cell of the invention, such as a prokaryotic or eukaryotichost cell in culture, can be used to produce (i.e., express) cytidinedeaminase-like protein. Accordingly, the invention further providesmethods for producing cytidine deaminase-like protein using the hostcells of the invention. In one embodiment, the method comprisesculturing the host cell of the invention, into which a recombinantexpression vector encoding an cytidine deaminase-like protein has beenintroduced, in a suitable medium such that cytidine deaminase-likeprotein is produced. In another embodiment, the method further comprisesisolating cytidine deaminase-like protein from the medium or the hostcell.

[0152] The host cells of the invention can also be used to producenonhuman transgenic animals. For example, in one embodiment, a host cellof the invention is a fertilized oocyte or an embryonic stem cell intowhich cytidine deaminase-like-coding sequences have been introduced.Such host cells can then be used to create nonhuman transgenic animalsin which exogenous cytidine deaminase-like sequences have beenintroduced into their genome or homologous recombinant animals in whichendogenous cytidine deaminase-like sequences have been altered. Suchanimals are useful for studying the function and/or activity of cytidinedeaminase-like genes and proteins and for identifying and/or evaluatingmodulators of cytidine deaminase-like activity. As used herein, a“transgenic animal” is a nonhuman animal, preferably a mammal, morepreferably a rodent such as a rat or mouse, in which one or more of thecells of the animal includes a transgene. Other examples of transgenicanimals include nonhuman primates, sheep, dogs, cows, goats, chickens,amphibians, etc. A transgene is exogenous DNA that is integrated intothe genome of a cell from which a transgenic animal develops and whichremains in the genome of the mature animal, thereby directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic animal. As used herein, a “homologousrecombinant animal” is a nonhuman animal, preferably a mammal, morepreferably a mouse, in which an endogenous cytidine deaminase-like genehas been altered by homologous recombination between the endogenous geneand an exogenous DNA molecule introduced into a cell of the animal,e.g., an embryonic cell of the animal, prior to development of theanimal.

[0153] A transgenic animal of the invention can be created byintroducing cytidine deaminase-like-encoding nucleic acid into the malepronuclei of a fertilized oocyte, e.g., by microinjection, retroviralinfection, and allowing the oocyte to develop in a pseudopregnant femalefoster animal. The cytidine deaminase-like cDNA sequence can beintroduced as a transgene into the genome of a nonhuman animal.Alternatively, a homologue of the mouse cytidine deaminase-like gene canbe isolated based on hybridization and used as a transgene. Intronicsequences and polyadenylation signals can also be included in thetransgene to increase the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to thecytidine deaminase-like transgene to direct expression of cytidinedeaminase-like protein to particular cells. Methods for generatingtransgenic animals via embryo manipulation and microinjection,particularly animals such as mice, have become conventional in the artand are described, for example, in U.S. Pat. Nos. 4,736,866, 4,870,009,and 4,873,191 and in Hogan (1986) Manipulating the Mouse Embryo (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similarmethods are used for production of other transgenic animals. Atransgenic founder animal can be identified based upon the presence ofthe cytidine deaminase-like transgene in its genome and/or expression ofcytidine deaminase-like mRNA in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding cytidine deaminase-like gene can further be bred toother transgenic animals carrying other transgenes.

[0154] To create a homologous recombinant animal, one prepares a vectorcontaining at least a portion of an cytidine deaminase-like gene or ahomolog of the gene into which a deletion, addition, or substitution hasbeen introduced to thereby alter, e.g., functionally disrupt, thecytidine deaminase-like gene. In a preferred embodiment, the vector isdesigned such that, upon homologous recombination, the endogenouscytidine deaminase-like gene is functionally disrupted (i.e., no longerencodes a functional protein; also referred to as a “knock out” vector).Alternatively, the vector can be designed such that, upon homologousrecombination, the endogenous cytidine deaminase-like gene is mutated orotherwise altered but still encodes functional protein (e.g., theupstream regulatory region can be altered to thereby alter theexpression of the endogenous cytidine deaminase-like protein). In thehomologous recombination vector, the altered portion of the cytidinedeaminase-like gene is flanked at its 5′ and 3′ ends by additionalnucleic acid of the cytidine deaminase-like gene to allow for homologousrecombination to occur between the exogenous cytidine deaminase-likegene carried by the vector and an endogenous cytidine deaminase-likegene in an embryonic stem cell. The additional flanking cytidinedeaminase-like nucleic acid is of sufficient length for successfulhomologous recombination with the endogenous gene. Typically, severalkilobases of flanking DNA (at both the 5′ and 3′ ends) are included inthe vector (see, e.g., Thomas and Capecchi (1987) Cell 51:503 for adescription of homologous recombination vectors). The vector isintroduced into an embryonic stem cell line (e.g., by electroporation),and cells in which the introduced cytidine deaminase-like gene hashomologously recombined with the endogenous cytidine deaminase-like geneare selected (see, e.g., Li et al. (1992) Cell 69:915). The selectedcells are then injected into a blastocyst of an animal (e.g., a mouse)to form aggregation chimeras (see, e.g., Bradley (1987) inTeratocarcinomas and Embryonic Stem Cells: A Practical Approach, ed.Robertson (IRL, Oxford pp. 113-152). A chimeric embryo can then beimplanted into a suitable pseudopregnant female foster animal and theembryo brought to term. Progeny harboring the homologously recombinedDNA in their germ cells can be used to breed animals in which all cellsof the animal contain the homologously recombined DNA by germlinetransmission of the transgene. Methods for constructing homologousrecombination vectors and homologous recombinant animals are describedfurther in Bradley (1991) Current Opinion in Bio/Technology 2:823-829and in PCT Publication Nos. WO 90/11354, WO 91/01140, WO 92/0968, and WO93/04169.

[0155] In another embodiment, transgenic nonhuman animals containingselected systems that allow for regulated expression of the transgenecan be produced. One example of such a system is the cre/loxPrecombinase system of bacteriophage P1. For a description of thecre/loxP recombinase system, see, e.g., Lakso et al. (1992) Proc. Natl.Acad. Sci. USA 89:6232-6236. Another example of a recombinase system isthe FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355). If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0156] Clones of the nonhuman transgenic animals described herein canalso be produced according to the methods described in Wilmut et al.(1997) Nature 385:810-813 and PCT Publication Nos. WO 97/07668 and WO97/07669.

[0157] IV. Pharmaceutical Compositions

[0158] The cytidine deaminase-like nucleic acid molecules, cytidinedeaminase-like proteins, and anti-cytidine deaminase-like antibodies(also referred to herein as “active compounds”) of the invention can beincorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the nucleic acidmolecule, protein, or antibody and a pharmaceutically acceptablecarrier. As used herein the language “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

[0159] The compositions of the invention are useful to treat any of thedisorders discussed herein. The compositions are provided intherapeutically effective amounts. By “therapeutically effectiveamounts” is intended an amount sufficient to modulate the desiredresponse. As defined herein, a therapeutically effective amount ofprotein or polypeptide (i.e., an effective dosage) ranges from about0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg bodyweight, more preferably about 0.1 to 20 mg/kg body weight, and even morepreferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7mg/kg, or 5 to 6 mg/kg body weight.

[0160] The skilled artisan will appreciate that certain factors mayinfluence the dosage required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a protein, polypeptide, or antibodycan include a single treatment or, preferably, can include a series oftreatments. In a preferred example, a subject is treated with antibody,protein, or polypeptide in the range of between about 0.1 to 20 mg/kgbody weight, one time per week for between about 1 to 10 weeks,preferably between 2 to 8 weeks, more preferably between about 3 to 7weeks, and even more preferably for about 4, 5, or 6 weeks. It will alsobe appreciated that the effective dosage of antibody, protein, orpolypeptide used for treatment may increase or decrease over the courseof a particular treatment. Changes in dosage may result and becomeapparent from the results of diagnostic assays as described herein.

[0161] The present invention encompasses agents which modulateexpression or activity. An agent may, for example, be a small molecule.For example, such small molecules include, but are not limited to,peptides, peptidomimetics, amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic or inorganic compounds (i.e., including heteroorganicand organometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds.

[0162] It is understood that appropriate doses of small molecule agentsdepends upon a number of factors within the knowledge of the ordinarilyskilled physician, veterinarian, or researcher. The dose(s) of the smallmolecule will vary, for example, depending upon the identity, size, andcondition of the subject or sample being treated, further depending uponthe route by which the composition is to be administered, if applicable,and the effect which the practitioner desires the small molecule to haveupon the nucleic acid or polypeptide of the invention. Exemplary dosesinclude milligram or microgram amounts of the small molecule perkilogram of subject or sample weight (e.g., about 1 microgram perkilogram to about 500 milligrams per kilogram, about 100 micrograms perkilogram to about 5 milligrams per kilogram, or about 1 microgram perkilogram to about 50 micrograms per kilogram. It is furthermoreunderstood that appropriate doses of a small molecule depend upon thepotency of the small molecule with respect to the expression or activityto be modulated. Such appropriate doses may be determined using theassays described herein. When one or more of these small molecules is tobe administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

[0163] A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes, or multiple dose vials made of glass or plastic.

[0164] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF; Parsippany, N.J.), or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion, and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

[0165] Sterile injectable solutions can be prepared by incorporating theactive compound (e.g., an cytidine deaminase-like protein oranti-cytidine deaminase-like antibody) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle that contains a basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying,which yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

[0166] Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth, or gelatin; an excipientsuch as starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. For administrationby inhalation, the compounds are delivered in the form of an aerosolspray from a pressurized container or dispenser that contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0167] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art. Thecompounds can also be prepared in the form of suppositories (e.g., withconventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

[0168] In one embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0169] It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated with each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. Depending on thetype and severity of the disease, about 1 μg/kg to about 15 mg/kg (e.g.,0.1 to 20 mg/kg) of antibody is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to about 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays. Anexemplary dosing regimen is disclosed in WO 94/04188. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

[0170] The nucleic acid molecules of the invention can be inserted intovectors and used as gene therapy vectors. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (U.S. Pat. No. 5,328,470), or by stereotactic injection(see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057).The pharmaceutical preparation of the gene therapy vector can includethe gene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

[0171] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0172] V. Uses and Methods of the Invention

[0173] The nucleic acid molecules, proteins, protein homologues, andantibodies described herein can be used in one or more of the followingmethods: (a) screening assays; (b) detection assays (e.g., chromosomalmapping, tissue typing, forensic biology); (c) predictive medicine(e.g., diagnostic assays, prognostic assays, monitoring clinical trials,and pharmacogenomics); and (d) methods of treatment (e.g., therapeuticand prophylactic). The isolated nucleic acid molecules of the inventioncan be used to express cytidine deaminase-like protein (e.g., via arecombinant expression vector in a host cell in gene therapyapplications), to detect cytidine deaminase-like mRNA (e.g., in abiological sample) or a genetic lesion in an cytidine deaminase-likegene, and to modulate cytidine deaminase-like activity. In addition, thecytidine deaminase-like proteins can be used to screen drugs orcompounds that modulate conversion of cytidine and deoxycytidine touridine and deoxyuridine, respectively, as well as to treat disorderscharacterized by insufficient or excessive production of cytidinedeaminase-like protein or production of cytidine deaminase-like proteinforms that have decreased or aberrant activity compared to cytidinedeaminase-like wild type protein. In addition, the anti-cytidinedeaminase-like antibodies of the invention can be used to detect andisolate cytidine deaminase-like proteins and modulate cytidinedeaminase-like activity.

[0174] A. Screening Assays

[0175] The invention provides a method (also referred to herein as a“screening assay”) for identifying modulators, i.e., candidate or testcompounds or agents (e.g., peptides, peptidomimetics, small molecules,or other drugs) that bind to cytidine deaminase-like proteins or have astimulatory or inhibitory effect on, for example, cytidinedeaminase-like expression or cytidine deaminase-like activity.

[0176] The test compounds of the present invention can be obtained usingany of the numerous approaches in combinatorial library methods known inthe art, including biological libraries, spatially addressable parallelsolid phase or solution phase libraries, synthetic library methodsrequiring deconvolution, the “one-bead one-compound” library method, andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, nonpeptide oligomer, orsmall molecule libraries of compounds (Lam (1997) Anticancer Drug Des.12:145).

[0177] Examples of methods for the synthesis of molecular libraries canbe found in the art, for example in: DeWitt et al. (1993) Proc. Natl.Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and Gallop et al. (1994) J. Med. Chem. 37:1233.

[0178] Libraries of compounds may be presented in solution (e.g.,Houghten (1992) Bio/Techniques 13:412-421), or on beads (Lam (1991)Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria(U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484;and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA89:1865-1869), or phage (Scott and Smith (1990) Science 249:386-390;Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl.Acad. Sci. USA 87:6378-6382; and Felici (1991) J. Mol. Biol.222:301-310).

[0179] Determining the ability of the test compound to bind to thecytidine deaminase-like protein can be accomplished, for example, bycoupling the test compound with a radioisotope or enzymatic label suchthat binding of the test compound to the cytidine deaminase-like proteinor biologically active portion thereof can be determined by detectingthe labeled compound in a complex. For example, test compounds can belabeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, andthe radioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, test compounds can beenzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

[0180] In a similar manner, one may determine the ability of thecytidine deaminase-like protein to bind to or interact with an cytidinedeaminase-like target molecule. By “target molecule” is intended amolecule with which an cytidine deaminase-like protein binds orinteracts in nature. In a preferred embodiment, the ability of thecytidine deaminase-like protein to bind to or interact with an cytidinedeaminase-like target molecule can be determined by monitoring theactivity of the target molecule. For example, the activity of the targetmolecule can be monitored by detecting a cytidine deamination event(e.g., such as in an mRNA editing, etc.), detecting catalytic/enzymaticactivity of the target on an appropriate substrate, detecting theinduction of a reporter gene (e.g., an cytidinedeaminase-like-responsive regulatory element operably linked to anucleic acid encoding a detectable marker, e.g. luciferase), ordetecting a cellular response, for example, decreased cell toxicity inresponse to a nucleoside analog.

[0181] In yet another embodiment, an assay of the present invention is acell-free assay comprising contacting an cytidine deaminase-like proteinor biologically active portion thereof with a test compound anddetermining the ability of the test compound to bind to the cytidinedeaminase-like protein or biologically active portion thereof. Bindingof the test compound to the cytidine deaminase-like protein can bedetermined either directly or indirectly as described above. In apreferred embodiment, the assay includes contacting the cytidinedeaminase-like protein or biologically active portion thereof with aknown compound that binds cytidine deaminase-like protein to form anassay mixture, contacting the assay mixture with a test compound, anddetermining the ability of the test compound to preferentially bind tocytidine deaminase-like protein or biologically active portion thereofas compared to the known compound.

[0182] In another embodiment, an assay is a cell-free assay comprisingcontacting cytidine deaminase-like protein or biologically activeportion thereof with a test compound and determining the ability of thetest compound to modulate (e.g., stimulate or inhibit) the activity ofthe cytidine deaminase-like protein or biologically active portionthereof. Determining the ability of the test compound to modulate theactivity of an cytidine deaminase-like protein can be accomplished, forexample, by determining the ability of the cytidine deaminase-likeprotein to bind to an cytidine deaminase-like target molecule asdescribed above for determining direct binding. In an alternativeembodiment, determining the ability of the test compound to modulate theactivity of an cytidine deaminase-like protein can be accomplished bydetermining the ability of the cytidine deaminase-like protein tofurther modulate an cytidine deaminase-like target molecule. Forexample, the catalytic/enzymatic activity of the target molecule on anappropriate substrate can be determined as previously described.

[0183] In yet another embodiment, the cell-free assay comprisescontacting the cytidine deaminase-like protein or biologically activeportion thereof with a known compound that binds an cytidinedeaminase-like protein to form an assay mixture, contacting the assaymixture with a test compound, and determining the ability of the testcompound to preferentially bind to or modulate the activity of ancytidine deaminase-like target molecule.

[0184] In the above-mentioned assays, it may be desirable to immobilizeeither an cytidine deaminase-like protein or its target molecule tofacilitate separation of complexed from uncomplexed forms of one or bothof the proteins, as well as to accommodate automation of the assay. Inone embodiment, a fusion protein can be provided that adds a domain thatallows one or both of the proteins to be bound to a matrix. For example,glutathione-S-transferase/cytidine deaminase-like fusion proteins orglutathione-S-transferase/target fusion proteins can be absorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione-derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the nonabsorbed targetprotein or cytidine deaminase-like protein, and the mixture incubatedunder conditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components andcomplex formation is measured either directly or indirectly, forexample, as described above. Alternatively, the complexes can bedissociated from the matrix, and the level of cytidine deaminase-likebinding or activity determined using standard techniques.

[0185] Other techniques for immobilizing proteins on matrices can alsobe used in the screening assays of the invention. For example, eithercytidine deaminase-like protein or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin.Biotinylated cytidine deaminase-like molecules or target molecules canbe prepared from biotin-NHS(N-hydroxy-succinimide) using techniques wellknown in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,Ill.), and immobilized in the wells of streptavidin-coated 96-wellplates (Pierce Chemicals). Alternatively, antibodies reactive with ancytidine deaminase-like protein or target molecules but which do notinterfere with binding of the cytidine deaminase-like protein to itstarget molecule can be derivatized to the wells of the plate, andunbound target or cytidine deaminase-like protein trapped in the wellsby antibody conjugation. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with thecytidine deaminase-like protein or target molecule, as well asenzyme-linked assays that rely on detecting an enzymatic activityassociated with the cytidine deaminase-like protein or target molecule.

[0186] In another embodiment, modulators of cytidine deaminase-likeexpression are identified in a method in which a cell is contacted witha candidate compound and the expression of cytidine deaminase-like mRNAor protein in the cell is determined relative to expression of cytidinedeaminase-like mRNA or protein in a cell in the absence of the candidatecompound. When expression is greater (statistically significantlygreater) in the presence of the candidate compound than in its absence,the candidate compound is identified as a stimulator of cytidinedeaminase-like mRNA or protein expression. Alternatively, whenexpression is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound isidentified as an inhibitor of cytidine deaminase-like mRNA or proteinexpression. The level of cytidine deaminase-like mRNA or proteinexpression in the cells can be determined by methods described hereinfor detecting cytidine deaminase-like mRNA or protein.

[0187] In yet another aspect of the invention, the cytidinedeaminase-like proteins can be used as “bait proteins” in a two-hybridassay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervoset al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.268:12046-12054; Bartel et al. (1993) Bio/Techniques 14:920-924;Iwabuchi et al. (1993) Oncogene 8:1693-1696; and PCT Publication No. WO94/10300), to identify other proteins, which bind to or interact withcytidine deaminase-like protein (“cytidine deaminase-like-bindingproteins” or “cytidine deaminase-like-bp”) and modulate cytidinedeaminase-like activity. Such cytidine deaminase-like-binding proteinsare also likely to be involved in the propagation of signals by thecytidine deaminase-like proteins as, for example, upstream or downstreamelements of the cytidine deaminase-like pathway.

[0188] This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

[0189] B. Detection Assays

[0190] Portions or fragments of the cDNA sequences identified herein(and the corresponding complete gene sequences) can be used in numerousways as polynucleotide reagents. For example, these sequences can beused to: (1) map their respective genes on a chromosome; (2) identify anindividual from a minute biological sample (tissue typing); and (3) aidin forensic identification of a biological sample. These applicationsare described in the subsections below.

[0191] 1. Chromosome Mapping

[0192] The isolated complete or partial cytidine deaminase-like genesequences of the invention can be used to map their respective cytidinedeaminase-like genes on a chromosome, thereby facilitating the locationof gene regions associated with genetic disease. Computer analysis ofcytidine deaminase-like sequences can be used to rapidly select PCRprimers (preferably 15-25 bp in length) that do not span more than oneexon in the genomic DNA, thereby simplifying the amplification process.These primers can then be used for PCR screening of somatic cell hybridscontaining individual human chromosomes. Only those hybrids containingthe human gene corresponding to the cytidine deaminase-like sequenceswill yield an amplified fragment.

[0193] Somatic cell hybrids are prepared by fusing somatic cells fromdifferent mammals (e.g., human and mouse cells). As hybrids of human andmouse cells grow and divide, they gradually lose human chromosomes inrandom order, but retain the mouse chromosomes. By using media in whichmouse cells cannot grow (because they lack a particular enzyme), but inwhich human cells can, the one human chromosome that contains the geneencoding the needed enzyme will be retained. By using various media,panels of hybrid cell lines can be established. Each cell line in apanel contains either a single human chromosome or a small number ofhuman chromosomes, and a full set of mouse chromosomes, allowing easymapping of individual genes to specific human chromosomes (D'Eustachioet al. (1983) Science 220:919-924). Somatic cell hybrids containing onlyfragments of human chromosomes can also be produced by using humanchromosomes with translocations and deletions.

[0194] Other mapping strategies that can similarly be used to map ancytidine deaminase-like sequence to its chromosome include in situhybridization (described in Fan et al. (1990) Proc. Natl. Acad. Sci. USA87:6223-27), pre-screening with labeled flow-sorted chromosomes, andpre-selection by hybridization to chromosome specific cDNA libraries.Furthermore, fluorescence in situ hybridization (FISH) of a DNA sequenceto a metaphase chromosomal spread can be used to provide a precisechromosomal location in one step. For a review of this technique, seeVerma eta a. (1988) Human Chromosomes: A Manual of Basic Techniques(Pergamon Press, NY). The FISH technique can be used with a DNA sequenceas short as 500 or 600 bases. However, clones larger than 1,000 baseshave a higher likelihood of binding to a unique chromosomal locationwith sufficient signal intensity for simple detection. Preferably 1,000bases, and more preferably 2,000 bases will suffice to get good resultsin a reasonable amount of time.

[0195] Reagents for chromosome mapping can be used individually to marka single chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

[0196] Another strategy to map the chromosomal location of cytidinedeaminase-like genes uses cytidine deaminase-like polypeptides andfragments and sequences of the present invention and antibodies specificthereto. This mapping can be carried out by specifically detecting thepresence of a cytidine deaminase-like polypeptide in members of a panelof somatic cell hybrids between cells of a first species of animal fromwhich the protein originates and cells from a second species of animal,and then determining which somatic cell hybrid(s) expresses thepolypeptide and noting the chromosomes(s) from the first species ofanimal that it contains. For examples of this technique, see Pajunen etal. (1988) Cytogenet. Cell. Genet. 47:37-41 and Van Keuren et al. (1986)Hum. Genet. 74:34-40. Alternatively, the presence of a cytidinedeaminase-like polypeptide in the somatic cell hybrids can be determinedby assaying an activity or property of the polypeptide, for example,enzymatic activity, as described in Bordelon-Riser et al. (1979) SomaticCell Genetics 5:597-613 and Owerbach et al. (1978) Proc. Natl. Acad.Sci. USA 75:5640-5644.

[0197] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. (Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man, available on-line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and disease, mapped to the same chromosomal region, canthen be identified through linkage analysis (co-inheritance ofphysically adjacent genes), described in, e.g., Egeland et al. (1987)Nature 325:783-787.

[0198] Moreover, differences in the DNA sequences between individualsaffected and unaffected with a disease associated with the cytidinedeaminase-like gene can be determined. If a mutation is observed in someor all of the affected individuals but not in any unaffectedindividuals, then the mutation is likely to be the causative agent ofthe particular disease. Comparison of affected and unaffectedindividuals generally involves first looking for structural alterationsin the chromosomes such as deletions or translocations that are visiblefrom chromosome spreads or detectable using PCR based on that DNAsequence. Ultimately, complete sequencing of genes from severalindividuals can be performed to confirm the presence of a mutation andto distinguish mutations from polymorphisms.

[0199] 2. Tissue Typing

[0200] The cytidine deaminase-like sequences of the present inventioncan also be used to identify individuals from minute biological samples.The United States military, for example, is considering the use ofrestriction fragment length polymorphism (RFLP) for identification ofits personnel. In this technique, an individual's genomic DNA isdigested with one or more restriction enzymes and probed on a Southernblot to yield unique bands for identification. The sequences of thepresent invention are useful as additional DNA markers for RFLP(described, e.g., in U.S. Pat. No. 5,272,057).

[0201] Furthermore, the sequences of the present invention can be usedto provide an alternative technique for determining the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the cytidine deaminase-like sequences of the invention canbe used to prepare two PCR primers from the 5′ and 3′ ends of thesequences. These primers can then be used to amplify an individual's DNAand subsequently sequence it.

[0202] Panels of corresponding DNA sequences from individuals, preparedin this manner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The cytidine deaminase-like sequences of the inventionuniquely represent portions of the human genome. Allelic variationoccurs to some degree in the coding regions of these sequences, and to agreater degree in the noncoding regions. It is estimated that allelicvariation between individual humans occurs with a frequency of aboutonce per each 500 bases. Each of the sequences described herein can, tosome degree, be used as a standard against which DNA from an individualcan be compared for identification purposes. The noncoding sequences ofSEQ ID NO:1 or 3 can comfortably provide positive individualidentification with a panel of perhaps 10 to 1,000 primers that eachyield a noncoding amplified sequence of 100 bases. If a predicted codingsequence, such as that in SEQ ID NO:2, is used, a more appropriatenumber of primers for positive individual identification would be 500 to2,000.

[0203] 3. Use of Partial Cytidine Deaminase-like Sequences in ForensicBiology

[0204] DNA-based identification techniques can also be used in forensicbiology. In this manner, PCR technology can be used to amplify DNAsequences taken from very small biological samples such as tissues,e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen foundat a crime scene. The amplified sequence can then be compared to astandard, thereby allowing identification of the origin of thebiological sample.

[0205] The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” that is unique to a particular individual. Asmentioned above, actual base sequence information can be used foridentification as an accurate alternative to patterns formed byrestriction enzyme generated fragments. Sequences targeted to noncodingregions of SEQ ID NO:1 or 3 are particularly appropriate for this use asgreater numbers of polymorphisms occur in the noncoding regions, makingit easier to differentiate individuals using this technique. Examples ofpolynucleotide reagents include the cytidine deaminase-like sequences orportions thereof, e.g., fragments derived from the noncoding regions ofSEQ ID NO:1 having a length of at least 20 or 30 bases.

[0206] The cytidine deaminase-like sequences described herein canfurther be used to provide polynucleotide reagents, e.g., labeled orlabelable probes that can be used in, for example, an in situhybridization technique, to identify a specific tissue. This can be veryuseful in cases where a forensic pathologist is presented with a tissueof unknown origin. Panels of such cytidine deaminase-like probes, can beused to identify tissue by species and/or by organ type.

[0207] In a similar fashion, these reagents, e.g., cytidinedeaminase-like primers or probes can be used to screen tissue culturefor contamination (i.e., screen for the presence of a mixture ofdifferent types of cells in a culture).

[0208] C. Predictive Medicine

[0209] The present invention also pertains to the field of predictivemedicine in which diagnostic assays, prognostic assays,pharmacogenomics, and monitoring clinical trails are used for prognostic(predictive) purposes to thereby treat an individual prophylactically.These applications are described in the subsections below.

[0210] 1. Diagnostic Assays

[0211] One aspect of the present invention relates to diagnostic assaysfor detecting cytidine deaminase-like protein and/or nucleic acidexpression as well as cytidine deaminase-like activity, in the contextof a biological sample. An exemplary method for detecting the presenceor absence of cytidine deaminase-like proteins in a biological sampleinvolves obtaining a biological sample from a test subject andcontacting the biological sample with a compound or an agent capable ofdetecting cytidine deaminase-like protein or nucleic acid (e.g., mRNA,genomic DNA) that encodes cytidine deaminase-like protein such that thepresence of cytidine deaminase-like protein is detected in thebiological sample. Results obtained with a biological sample from thetest subject may be compared to results obtained with a biologicalsample from a control subject.

[0212] “Misexpression or aberrant expression”, as used herein, refers toa non-wild type pattern of gene expression, at the RNA or protein level.It includes: expression at non-wild type levels, i.e., over or underexpression; a pattern of expression that differs from wild type in termsof the time or stage at which the gene is expressed, e.g., increased ordecreased expression (as compared with wild type) at a predetermineddevelopmental period or stage; a pattern of expression that differs fromwild type in terms of decreased expression (as compared with wild type)in a predetermined cell type or tissue type; a pattern of expressionthat differs from wild type in terms of the splicing size, amino acidsequence, post-transitional modification, or biological activity of theexpressed polypeptide; a pattern of expression that differs from wildtype in terms of the effect of an environmental stimulus orextracellular stimulus on expression of the gene, e.g., a pattern ofincreased or decreased expression (as compared with wild type) in thepresence of an increase or decrease in the strength of the stimulus.

[0213] A preferred agent for detecting cytidine deaminase-like mRNA orgenomic DNA is a labeled nucleic acid probe capable of hybridizing tocytidine deaminase-like mRNA or genomic DNA. The nucleic acid probe canbe, for example, a full-length cytidine deaminase-like nucleic acid,such as the nucleic acid of SEQ ID NO:1 or 3, or a portion thereof, suchas a nucleic acid molecule of at least 15, 30, 50, 100, 250, or 500nucleotides in length and sufficient to specifically hybridize understringent conditions to cytidine deaminase-like mRNA or genomic DNA.Other suitable probes for use in the diagnostic assays of the inventionare described herein.

[0214] A preferred agent for detecting cytidine deaminase-like proteinis an antibody capable of binding to cytidine deaminase-like protein,preferably an antibody with a detectable label. Antibodies can bepolyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof (e.g., Fab or F(abN)₂) can be used. The term “labeled”,with regard to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by coupling (i.e., physically linking)a detectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with another reagentthat is directly labeled. Examples of indirect labeling includedetection of a primary antibody using a fluorescently labeled secondaryantibody and end-labeling of a DNA probe with biotin such that it can bedetected with fluorescently labeled streptavidin.

[0215] The term “biological sample” is intended to include tissues,cells, and biological fluids isolated from a subject, as well astissues, cells, and fluids present within a subject. That is, thedetection method of the invention can be used to detect cytidinedeaminase-like mRNA, protein, or genomic DNA in a biological sample invitro as well as in vivo. For example, in vitro techniques for detectionof cytidine deaminase-like mRNA include Northern hybridizations and insitu hybridizations. In vitro techniques for detection of cytidinedeaminase-like protein include enzyme linked immunosorbent assays(ELISAs), Western blots, immunoprecipitations, and immunofluorescence.In vitro techniques for detection of cytidine deaminase-like genomic DNAinclude Southern hybridizations. Furthermore, in vivo techniques fordetection of cytidine deaminase-like protein include introducing into asubject a labeled anti-cytidine deaminase-like antibody. For example,the antibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

[0216] In one embodiment, the biological sample contains proteinmolecules from the test subject. Alternatively, the biological samplecan contain mRNA molecules from the test subject or genomic DNAmolecules from the test subject.

[0217] The invention also encompasses kits for detecting the presence ofcytidine deaminase-like proteins in a biological sample (a test sample).Such kits can be used to determine if a subject is suffering from or isat increased risk of developing a disorder associated with aberrantexpression of cytidine deaminase-like protein. For example, the kit cancomprise a labeled compound or agent capable of detecting cytidinedeaminase-like protein or mRNA in a biological sample and means fordetermining the amount of an cytidine deaminase-like protein in thesample (e.g., an anti-cytidine deaminase-like antibody or anoligonucleotide probe that binds to DNA encoding an cytidinedeaminase-like protein, e.g., SEQ ID NO:1 or 3). Kits can also includeinstructions for observing that the tested subject is suffering from oris at risk of developing a disorder associated with aberrant expressionof cytidine deaminase-like sequences if the amount of cytidinedeaminase-like protein or mRNA is above or below a normal level.

[0218] For antibody-based kits, the kit can comprise, for example: (1) afirst antibody (e.g., attached to a solid support) that binds tocytidine deaminase-like protein; and, optionally, (2) a second,different antibody that binds to cytidine deaminase-like protein or thefirst antibody and is conjugated to a detectable agent. Foroligonucleotide-based kits, the kit can comprise, for example: (1) anoligonucleotide, e.g., a detectably labeled oligonucleotide, thathybridizes to an cytidine deaminase-like nucleic acid sequence or (2) apair of primers useful for amplifying an cytidine deaminase-like nucleicacid molecule.

[0219] The kit can also comprise, e.g., a buffering agent, apreservative, or a protein stabilizing agent. The kit can also comprisecomponents necessary for detecting the detectable agent (e.g., an enzymeor a substrate). The kit can also contain a control sample or a seriesof control samples that can be assayed and compared to the test samplecontained. Each component of the kit is usually enclosed within anindividual container, and all of the various containers are within asingle package along with instructions for observing whether the testedsubject is suffering from or is at risk of developing a disorderassociated with aberrant expression of cytidine deaminase-like proteins.

[0220] 2. Other Diagnostic Assays

[0221] In another aspect, the invention features a method of analyzing aplurality of capture probes. The method can be used, e.g., to analyzegene expression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the plurality,and each address of the plurality having a unique capture probe, e.g., anucleic acid or peptide sequence; contacting the array with a cytidinedeaminase-like nucleic acid, preferably purified, polypeptide,preferably purified, or antibody, and thereby evaluating the pluralityof capture probes. Binding, e.g., in the case of a nucleic acid,hybridization, with a capture probe at an address of the plurality, isdetected, e.g., by signal generated from a label attached to thecytidine deaminase-like nucleic acid, polypeptide, or antibody. Thecapture probes can be a set of nucleic acids from a selected sample,e.g., a sample of nucleic acids derived from a control or non-stimulatedtissue or cell.

[0222] The method can include contacting the cytidine deaminase-likenucleic acid, polypeptide, or antibody with a first array having aplurality of capture probes and a second array having a differentplurality of capture probes. The results of each hybridization can becompared, e.g., to analyze differences in expression between a first andsecond sample. The first plurality of capture probes can be from acontrol sample, e.g., a wild type, normal, or non-diseased,non-stimulated, sample, e.g., a biological fluid, tissue, or cellsample. The second plurality of capture probes can be from anexperimental sample, e.g., a mutant type, at risk, disease-state ordisorder-state, or stimulated, sample, e.g., a biological fluid, tissue,or cell sample.

[0223] The plurality of capture probes can be a plurality of nucleicacid probes each of which specifically hybridizes, with an allele of acytidine deaminase-like sequence of the invention. Such methods can beused to diagnose a subject, e.g., to evaluate risk for a disease ordisorder, to evaluate suitability of a selected treatment for a subject,to evaluate whether a subject has a disease or disorder.

[0224] The method can be used to detect single nucleotide polymorphisms(SNPs), as described below.

[0225] In another aspect, the invention features a method of analyzing aplurality of probes. The method is useful, e.g., for analyzing geneexpression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the pluralityhaving a unique capture probe, e.g., wherein the capture probes are froma cell or subject which express a cytidine deaminase-like polypeptide ofthe invention or from a cell or subject in which a cytidinedeaminase-like-mediated response has been elicited, e.g., by contact ofthe cell with a cytidine deaminase-like nucleic acid or protein of theinvention, or administration to the cell or subject a cytidinedeaminase-like nucleic acid or protein of the invention; contacting thearray with one or more inquiry probes, wherein an inquiry probe can be anucleic acid, polypeptide, or antibody (which is preferably other than acytidine deaminase-like nucleic acid, polypeptide, or antibody of theinvention); providing a two dimensional array having a plurality ofaddresses, each address of the plurality being positionallydistinguishable from each other address of the plurality, and eachaddress of the plurality having a unique capture probe, e.g., whereinthe capture probes are from a cell or subject which does not express acytidine deaminase-like sequence of the invention (or does not expressas highly as in the case of the cytidine deaminase-like positiveplurality of capture probes) or from a cell or subject in which acytidine deaminase-like-mediated response has not been elicited (or hasbeen elicited to a lesser extent than in the first sample); contactingthe array with one or more inquiry probes (which is preferably otherthan a cytidine deaminase-like nucleic acid, polypeptide, or antibody ofthe invention), and thereby evaluating the plurality of capture probes.Binding, e.g., in the case of a nucleic acid, hybridization, with acapture probe at an address of the plurality, is detected, e.g., bysignal generated from a label attached to the nucleic acid, polypeptide,or antibody.

[0226] In another aspect, the invention features a method of analyzing acytidine deaminase-like sequence of the invention, e.g., analyzingstructure, function, or relatedness to other nucleic acid or amino acidsequences. The method includes: providing a cytidine deaminase-likenucleic acid or amino acid sequence, e.g., the 26934 sequence set forthin SEQ ID NO:1 or 3 or a portion thereof; comparing the cytidinedeaminase-like sequence with one or more preferably a plurality ofsequences from a collection of sequences, e.g., a nucleic acid orprotein sequence database; to thereby analyze the cytidinedeaminase-like sequence of the invention.

[0227] The method can include evaluating the sequence identity between acytidine deaminase-like sequence of the invention, e.g., the 26934sequence, and a database sequence. The method can be performed byaccessing the database at a second site, e.g., over the internet.

[0228] In another aspect, the invention features, a set ofoligonucleotides, useful, e.g., for identifying SNP's, or identifyingspecific alleles of a cytidine deaminase-like sequence of the invention,e.g., the 26934 sequence. The set includes a plurality ofoligonucleotides, each of which has a different nucleotide at aninterrogation position, e.g., an SNP or the site of a mutation. In apreferred embodiment, the oligonucleotides of the plurality identical insequence with one another (except for differences in length). Theoligonucleotides can be provided with differential labels, such that anoligonucleotides which hybridizes to one allele provides a signal thatis distinguishable from an oligonucleotides which hybridizes to a secondallele.

[0229] 3. Prognostic Assays

[0230] The methods described herein can furthermore be utilized asdiagnostic or prognostic assays to identify subjects having or at riskof developing a disease or disorder associated with cytidinedeaminase-like protein, cytidine deaminase-like nucleic acid expression,or cytidine deaminase-like activity. Prognostic assays can be used forprognostic or predictive purposes to thereby prophylactically treat anindividual prior to the onset of a disorder characterized by orassociated with cytidine deaminase-like protein, cytidine deaminase-likenucleic acid expression, or cytidine deaminase-like activity.

[0231] Thus, the present invention provides a method in which a testsample is obtained from a subject, and cytidine deaminase-like proteinor nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein thepresence of cytidine deaminase-like protein or nucleic acid isdiagnostic for a subject having or at risk of developing a disease ordisorder associated with aberrant cytidine deaminase-like expression oractivity. As used herein, a “test sample” refers to a biological sampleobtained from a subject of interest. For example, a test sample can be abiological fluid (e.g., serum), cell sample, or tissue.

[0232] Furthermore, using the prognostic assays described herein, thepresent invention provides methods for determining whether a subject canbe administered a specific agent (e.g., an agonist, antagonist,peptidomimetic, protein, peptide, nucleic acid, small molecule, or otherdrug candidate) or class of agents (e.g., agents of a type that decreasecytidine deaminase-like activity) to effectively treat a disease ordisorder associated with aberrant cytidine deaminase-like expression oractivity. In this manner, a test sample is obtained and cytidinedeaminase-like protein or nucleic acid is detected. The presence ofcytidine deaminase-like protein or nucleic acid is diagnostic for asubject that can be administered the agent to treat a disorderassociated with aberrant cytidine deaminase-like expression or activity.

[0233] The methods of the invention can also be used to detect geneticlesions or mutations in an cytidine deaminase-like gene, therebydetermining if a subject with the lesioned gene is at risk for adisorder characterized by aberrant cytidine deamination. In preferredembodiments, the methods include detecting, in a sample of cells fromthe subject, the presence or absence of a genetic lesion or mutationcharacterized by at least one of an alteration affecting the integrityof a gene encoding an cytidine deaminase-like-protein, or themisexpression of the cytidine deaminase-like gene. For example, suchgenetic lesions or mutations can be detected by ascertaining theexistence of at least one of: (1) a deletion of one or more nucleotidesfrom an cytidine deaminase-like gene; (2) an addition of one or morenucleotides to an cytidine deaminase-like gene; (3) a substitution ofone or more nucleotides of an cytidine deaminase-like gene; (4) achromosomal rearrangement of an cytidine deaminase-like gene; (5) analteration in the level of a messenger RNA transcript of an cytidinedeaminase-like gene; (6) an aberrant modification of an cytidinedeaminase-like gene, such as of the methylation pattern of the genomicDNA; (7) the presence of a non-wild-type splicing pattern of a messengerRNA transcript of an cytidine deaminase-like gene; (8) a non-wild-typelevel of an cytidine deaminase-like-protein; (9) an allelic loss of ancytidine deaminase-like gene; and (10) an inappropriatepost-translational modification of an cytidine deaminase-like-protein.As described herein, there are a large number of assay techniques knownin the art that can be used for detecting lesions in an cytidinedeaminase-like gene. Any cell type or tissue, in which cytidinedeaminase-like proteins are expressed may be utilized in the prognosticassays described herein.

[0234] In certain embodiments, detection of the lesion involves the useof a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in the cytidinedeaminase-like-gene (see, e.g., Abravaya et al. (1995) Nucleic AcidsRes. 23:675-682). It is anticipated that PCR and/or LCR may be desirableto use as a preliminary amplification step in conjunction with any ofthe techniques used for detecting mutations described herein.

[0235] Alternative amplification methods include self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

[0236] In an alternative embodiment, mutations in an cytidinedeaminase-like gene from a sample cell can be identified by alterationsin restriction enzyme cleavage patterns of isolated test sample andcontrol DNA digested with one or more restriction endonucleases.Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Pat.No. 5,498,531) can be used to score for the presence of specificmutations by development or loss of a ribozyme cleavage site.

[0237] In other embodiments, genetic mutations in an cytidinedeaminase-like molecule can be identified by hybridizing a sample andcontrol nucleic acids, e.g., DNA or RNA, to high density arrayscontaining hundreds or thousands of oligonucleotides probes (Cronin etal. (1996) Human Mutation 7:244-255; Kozal et al. (1996) Nature Medicine2:753-759). In yet another embodiment, any of a variety of sequencingreactions known in the art can be used to directly sequence the cytidinedeaminase-like gene and detect mutations by comparing the sequence ofthe sample cytidine deaminase-like gene with the corresponding wild-type(control) sequence. Examples of sequencing reactions include those basedon techniques developed by Maxim and Gilbert ((1977) Proc. Natl. Acad.Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463).It is also contemplated that any of a variety of automated sequencingprocedures can be utilized when performing the diagnostic assays ((1995)Bio/Techniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT Publication No. WO 94/16101; Cohen et al. (1996) Adv.Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem.Biotechnol. 38:147-159).

[0238] Other methods for detecting mutations in the cytidinedeaminase-like gene include methods in which protection from cleavageagents is used to detect mismatched bases in RNA/RNA or RNA/DNAheteroduplexes (Myers et al. (1985) Science 230:1242). See, also Cottonet al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992)Methods Enzymol. 217:286-295. In a preferred embodiment, the control DNAor RNA can be labeled for detection.

[0239] In still another embodiment, the mismatch cleavage reactionemploys one or more “DNA mismatch repair” enzymes that recognizemismatched base pairs in double-stranded DNA in defined systems fordetecting and mapping point mutations in cytidine deaminase-like cDNAsobtained from samples of cells. See, e.g., Hsu et al. (1994)Carcinogenesis 15:1657-1662. According to an exemplary embodiment, aprobe based on an cytidine deaminase-like sequence, e.g., a wild-typecytidine deaminase-like sequence, is hybridized to a cDNA or other DNAproduct from a test cell(s). The duplex is treated with a DNA mismatchrepair enzyme, and the cleavage products, if any, can be detected fromelectrophoresis protocols or the like. See, e.g., U.S. Pat. No.5,459,039.

[0240] In other embodiments, alterations in electrophoretic mobilitywill be used to identify mutations in cytidine deaminase-like genes. Forexample, single-strand conformation polymorphism (SSCP) may be used todetect differences in electrophoretic mobility between mutant andwild-type nucleic acids (Orita et al. (1989) Proc. Natl. Acad. Sci. USA86:2766; see also Cotton (1993) Mutat. Res. 285:125-144; Hayashi (1992)Genet. Anal. Tech. Appl. 9:73-79). The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double-stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

[0241] In yet another embodiment, the movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (DGGE) (Myers etal. (1985) Nature 313:495). When DGGE is used as the method of analysis,DNA will be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

[0242] Examples of other techniques for detecting point mutationsinclude, but are not limited to, selective oligonucleotidehybridization, selective amplification, or selective primer extension.For example, oligonucleotide primers may be prepared in which the knownmutation is placed centrally and then hybridized to target DNA underconditions that permit hybridization only if a perfect match is found(Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl.Acad. Sci. USA 86:6230). Such allele-specific oligonucleotides arehybridized to PCR-amplified target DNA or a number of differentmutations when the oligonucleotides are attached to the hybridizingmembrane and hybridized with labeled target DNA.

[0243] Alternatively, allele-specific amplification technology, whichdepends on selective PCR amplification, may be used in conjunction withthe instant invention. Oligonucleotides used as primers for specificamplification may carry the mutation of interest in the center of themolecule so that amplification depends on differential hybridization(Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme3N end of one primer where, under appropriate conditions, mismatch canprevent or reduce polymerase extension (Prossner (1993) Tibtech 11:238).In addition, it may be desirable to introduce a novel restriction sitein the region of the mutation to create cleavage-based detection(Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated thatin certain embodiments amplification may also be performed using Taqligase for amplification (Barany (1991) Proc. Natl. Acad. Sci. USA88:189). In such cases, ligation will occur only if there is a perfectmatch at the 3N end of the 5N sequence making it possible to detect thepresence of a known mutation at a specific site by looking for thepresence or absence of amplification.

[0244] The methods described herein may be performed, for example, byutilizing prepackaged diagnostic kits comprising at least one probenucleic acid or antibody reagent described herein, which may beconveniently used, e.g., in clinical settings to diagnosed patientsexhibiting symptoms or family history of a disease or illness involvingan cytidine deaminase-like gene.

[0245] 4. Pharmacogenomics

[0246] Agents, or modulators that have a stimulatory or inhibitoryeffect on cytidine deaminase-like activity (e.g., cytidinedeaminase-like gene expression) as identified by a screening assaydescribed herein, can be administered to individuals to treat(prophylactically or therapeutically) disorders associated with aberrantcytidine deaminase-like activity. In conjunction with such treatment,the pharmacogenomics (i.e., the study of the relationship between anindividual's genotype and that individual's response to a foreigncompound or drug) of the individual may be considered. Differences inmetabolism of therapeutics can lead to severe toxicity or therapeuticfailure by altering the relation between dose and blood concentration ofthe pharmacologically active drug. Thus, the pharmacogenomics of theindividual permits the selection of effective agents (e.g., drugs) forprophylactic or therapeutic treatments based on a consideration of theindividual's genotype. Such pharmacogenomics can further be used todetermine appropriate dosages and therapeutic regimens. Accordingly, theactivity of cytidine deaminase-like protein, expression of cytidinedeaminase-like nucleic acid, or mutation content of cytidinedeaminase-like genes in an individual can be determined to therebyselect appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual.

[0247] Pharmacogenomics deals with clinically significant hereditaryvariations in the response to drugs due to altered drug disposition andabnormal action in affected persons. See, e.g., Linder (1997) Clin.Chem. 43(2):254-266. In general, two types of pharmacogenetic conditionscan be differentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body are referred to as “altered drugaction.” Genetic conditions transmitted as single factors altering theway the body acts on drugs are referred to as “altered drug metabolism”.These pharmacogenetic conditions can occur either as rare defects or aspolymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency(G6PD) is a common inherited enzymopathy in which the main clinicalcomplication is haemolysis after ingestion of oxidant drugs(antimalarials, sulfonamides, analgesics, nitrofurans) and consumptionof fava beans.

[0248] One pharmacogenomics approach to identifying genes that predictdrug response, known as “a genome-wide association”, relies primarily ona high-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, an “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

[0249] Alternatively, a method termed the “candidate gene approach”, canbe utilized to identify genes that predict drug response. According tothis method, if a gene that encodes a drug's target is known (e.g., acytidine deaminase-like protein of the present invention), all commonvariants of that gene can be fairly easily identified in the populationand it can be determined if having one version of the gene versusanother is associated with a particular drug response.

[0250] Alternatively, a method termed the “gene expression profiling”,can be utilized to identify genes that predict drug response. Forexample, the gene expression of an animal dosed with a drug (e.g., acytidine deaminase-like molecule or cytidine deaminase-like modulator ofthe present invention) can give an indication whether gene pathwaysrelated to toxicity have been turned on.

[0251] Information generated from more than one of the abovepharmacogenonics approaches can be used to determine appropriate dosageand treatment regimens for prophylactic or therapeutic treatment of anindividual. This knowledge, when applied to dosing or drug selection,can avoid adverse reactions or therapeutic failure and thus enhancetherapeutic or prophylactic efficiency when treating a subject with acytidine deaminase-like molecule or cytidine deaminase-like modulator ofthe invention, such as a modulator identified by one of the exemplaryscreening assays described herein.

[0252] The present invention further provides methods for identifyingnew agents, or combinations, that are based on identifying agents thatmodulate the activity of one or more of the gene products encoded by oneor more of the cytidine deaminase-like genes of the present invention,wherein these products may be associated with resistance of the cells toa therapeutic agent. Specifically, the activity of the proteins encodedby the cytidine deaminase-like genes of the present invention can beused as a basis for identifying agents for overcoming agent resistance.By blocking the activity of one or more of the resistance proteins,target cells, will become sensitive to treatment with an agent that theunmodified target cells were resistant to.

[0253] Monitoring the influence of agents (e.g., drugs) on theexpression or activity of a cytidine deaminase-like protein can beapplied in clinical trials. For example, the effectiveness of an agentdetermined by a screening assay as described herein to increase cytidinedeaminase-like gene expression, protein levels, or upregulate cytidinedeaminase-like activity, can be monitored in clinical trials of subjectsexhibiting decreased cytidine deaminase-like gene expression, proteinlevels, or down-regulated cytidine deaminase-like activity.Alternatively, the effectiveness of an agent determined by a screeningassay to decrease cytidine deaminase-like gene expression, proteinlevels, or downregulate cytidine deaminase-like activity, can bemonitored in clinical trials of subjects exhibiting increased cytidinedeaminase-like gene expression, protein levels, or upregulated cytidinedeaminase-like activity. In such clinical trials, the expression oractivity of a cytidine deaminase-like gene, and preferably, other genesthat have been implicated in, for example, a cytidinedeaminase-like-associated disorder can be used as a “read out” ormarkers of the phenotype of a particular cell.

[0254] As an illustrative embodiment, the activity of drug metabolizingenzymes is a major determinant of both the intensity and duration ofdrug action. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, a PM will show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

[0255] Thus, the activity of cytidine deaminase-like protein, expressionof cytidine deaminase-like nucleic acid, or mutation content of cytidinedeaminase-like genes in an individual can be determined to therebyselect appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual. In addition, pharmacogenetic studies can be used toapply genotyping of polymorphic alleles encoding drug-metabolizingenzymes to the identification of an individual's drug responsivenessphenotype. This knowledge, when applied to dosing or drug selection, canavoid adverse reactions or therapeutic failure and thus enhancetherapeutic or prophylactic efficiency when treating a subject with ancytidine deaminase-like modulator, such as a modulator identified by oneof the exemplary screening assays described herein.

[0256] 5. Monitoring of Effects During Clinical Trials

[0257] Monitoring the influence of agents (e.g., drugs, compounds) onthe expression or activity of cytidine deaminase-like genes can beapplied not only in basic drug screening but also in clinical trials.For example, the effectiveness of an agent, as determined by a screeningassay as described herein, to increase or decrease cytidinedeaminase-like gene expression, protein levels, or protein activity, canbe monitored in clinical trials of subjects exhibiting decreased orincreased cytidine deaminase-like gene expression, protein levels, orprotein activity. In such clinical trials, cytidine deaminase-likeexpression or activity can be used as a marker of the immuneresponsiveness of a particular cell.

[0258] For example, and not by way of limitation, genes that aremodulated in cells by treatment with an agent (e.g., compound, drug, orsmall molecule) that modulates cytidine deaminase-like activity (e.g.,as identified in a screening assay described herein) can be identified.Thus, to study the effect of agents on cellular disorders influenced bycytidine deaminase, for example, in a clinical trial, cells can beisolated and RNA prepared and analyzed for the levels of expression ofcytidine deaminase-like genes and other genes implicated in thedisorder. The levels of gene expression (i.e., a gene expressionpattern) can be quantified by Northern blot analysis or RT-PCR, asdescribed herein, or alternatively by measuring the amount of proteinproduced, by one of the methods as described herein, or by measuring thelevels of activity of cytidine deaminase-like genes or other genes. Inthis way, the gene expression pattern can serve as a marker, indicativeof the physiological response of the cells to the agent. Accordingly,this response state may be determined before, and at various pointsduring, treatment of the individual with the agent.

[0259] In a preferred embodiment, the present invention provides amethod for monitoring the effectiveness of treatment of a subject withan agent (e.g., an agonist, antagonist, peptidomimetic, protein,peptide, nucleic acid, small molecule, or other drug candidateidentified by the screening assays described herein) comprising thesteps of (1) obtaining a preadministration sample from a subject priorto administration of the agent; (2) detecting the level of expression ofan cytidine deaminase-like protein, mRNA, or genomic DNA in thepreadministration sample; (3) obtaining one or more postadministrationsamples from the subject; (4) detecting the level of expression oractivity of the cytidine deaminase-like protein, mRNA, or genomic DNA inthe postadministration samples; (5) comparing the level of expression oractivity of the cytidine deaminase-like protein, mRNA, or genomic DNA inthe preadministration sample with the cytidine deaminase-like protein,mRNA, or genomic DNA in the postadministration sample or samples; and(vi) altering the administration of the agent to the subject accordinglyto bring about the desired effect, i.e., for example, an increase or adecrease in the expression or activity of an cytidine deaminase-likeprotein.

[0260] C. Methods of Treatment

[0261] The present invention provides for both prophylactic andtherapeutic methods of treating a subject at risk of (or susceptible to)a disorder or having a disorder associated with aberrant cytidinedeaminase-like expression or activity. Additionally, the compositions ofthe invention find use in the treatment of disorders described herein.Thus, therapies for disorders associated with cytidine deaminase-likedisorder are encompassed herein. “Subject”, as used herein, can refer toa mammal, e.g., a human, or to an experimental or animal or diseasemodel. The subject can also be a non-human animal, e.g., a horse, cow,goat, or other domestic animal.

[0262] “Treatment” is herein defined as the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease or the predisposition toward disease. A“therapeutic agent” includes, but is not limited to, small molecules,peptides, antibodies, ribozymes and antisense oligonucleotides.

[0263] 1. Prophylactic Methods

[0264] In one aspect, the invention provides a method for preventing ina subject a disease or condition associated with an aberrant cytidinedeaminase-like expression or activity by administering to the subject anagent that modulates cytidine deaminase-like expression or at least onecytidine deaminase-like gene activity. Subjects at risk for a diseasethat is caused, or contributed to, by aberrant cytidine deaminase-likeexpression or activity can be identified by, for example, any or acombination of diagnostic or prognostic assays as described herein.Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of the cytidine deaminase-likeaberrancy, such that a disease or disorder is prevented or,alternatively, delayed in its progression. Depending on the type ofcytidine deaminase-like aberrancy, for example, an cytidinedeaminase-like agonist or cytidine deaminase-like antagonist agent canbe used for treating the subject. The appropriate agent can bedetermined based on screening assays described herein.

[0265] 2. Therapeutic Methods

[0266] Another aspect of the invention pertains to methods of modulatingcytidine deaminase-like expression or activity for therapeutic purposes.The modulatory method of the invention involves contacting a cell withan agent that modulates one or more of the activities of cytidinedeaminase-like protein activity associated with the cell. An agent thatmodulates cytidine deaminase-like protein activity can be an agent asdescribed herein, such as a nucleic acid or a protein, anaturally-occurring cognate ligand of an cytidine deaminase-likeprotein, a peptide, an cytidine deaminase-like peptidomimetic, or othersmall molecule. In one embodiment, the agent stimulates one or more ofthe biological activities of cytidine deaminase-like protein. Examplesof such stimulatory agents include active cytidine deaminase-likeprotein and a nucleic acid molecule encoding an cytidine deaminase-likeprotein that has been introduced into the cell. In another embodiment,the agent inhibits one or more of the biological activities of cytidinedeaminase-like protein. Examples of such inhibitory agents includeantisense cytidine deaminase-like nucleic acid molecules andanti-cytidine deaminase-like antibodies.

[0267] These modulatory methods can be performed in vitro (e.g., byculturing the cell with the agent) or, alternatively, in vivo (e.g., byadministering the agent to a subject). As such, the present inventionprovides methods of treating an individual afflicted with a disease ordisorder characterized by aberrant expression or activity of an cytidinedeaminase-like protein or nucleic acid molecule. In one embodiment, themethod involves administering an agent (e.g., an agent identified by ascreening assay described herein), or a combination of agents, thatmodulates (e.g., upregulates or down-regulates) cytidine deaminase-likeexpression or activity. In another embodiment, the method involvesadministering an cytidine deaminase-like protein or nucleic acidmolecule as therapy to compensate for reduced or aberrant cytidinedeaminase-like expression or activity.

[0268] Stimulation of cytidine deaminase-like activity is desirable insituations in which an cytidine deaminase-like protein is abnormallydownregulated and/or in which increased cytidine deaminase-like activityis likely to have a beneficial effect. Conversely, inhibition ofcytidine deaminase-like activity is desirable in situations in whichcytidine deaminase-like activity is abnormally upregulated and/or inwhich decreased cytidine deaminase-like activity is likely to have abeneficial effect.

[0269] This invention is further illustrated by the following examples,which should not be construed as limiting.

EXPERIMENTAL Example 1 Tissue Distribution of Cytidine Deaminase-LikemRNA

[0270] The 26934 clone was originally identified using a transcriptionalprofiling experiment comparing 3 normal ovarian epithelial (NOE) cellsto 2 clinical ovarian ascites samples. 26934 was overexpressed in theascites samples compared to the NOE cells. This same paradigm wasconfirmed via TaqMan expression analysis as shown in FIG. 6. Thequantitative RT-PCR (Reverse Transcriptase Polymerase Chain Reaction;Taqman® brand PCR kit, Applied Biosystems) was performed according tothe kit manufacturer's instructions.

[0271] TaqMan analysis of the 26934 sequence revealed expression in anumber of tissues. As shown in FIG. 4, TaqMan expression analysisindicates that the 26934 mRNA is abundantly expressed in normal artery,brain cortex and hypothalamus and moderately expressed in HUVEC cells,kidney, skeletal muscle, nerve, DRG, normal ovary, lung tumor anderythroid cells. Low expression can be noted in many other tissuesincluding normal heart, normal vein, diseased aorta, CHF heart,pancreas, primary osteoblasts, normal skin spinal cord, normal breast,breast tumor, ovarian tumor, normal prostate, prostate tumor, salivaryglands, normal colon, colon tumor, normal lung, lung COPD, IBD colon,normal liver, liver fibrosis, normal spleen, tonsil, and smallintestine.

[0272] In an expanded oncology based TaqMan panel shown in FIG. 5, 26934mRNA was expressed in normal breast, ovary, lung and colon tissues andprimary and metastatic tumors associated with all of these tissues.Expression was also noted in tumors of the cervix.

[0273] Additional TaqMan expression panels examining expression of the26934 mRNA in ovarian cell based models indicate this gene may beassociated with cMyc and growth factor expression. This was shown due tothe expression of 26934 mRNA at 3 hr and 6 hr after serum addition tothe HEY cells (FIGS. 7 and 10). This result correlates with cMycactivation in this model system.

[0274] Using another cell model, expression of 26934 mRNA was alsoinduced after addition of the growth factors EGF (epidermal growthfactor) and Hrg (Heregulin) to the ovarian cells SKOV-3, indicating thatthis gene may be responsive to growth factor stimulation. See FIGS. 8, 9and 10.

Example 2 Recombinant Expression of Cytidine Deaminase-Like in BacterialCells

[0275] In this example, the cytidine deaminase-like sequence isexpressed as a recombinant glutathione-S-transferase (GST) fusionpolypeptide in E. coli and the fusion polypeptide is isolated andcharacterized. Specifically, the sequence is fused to GST and thisfusion polypeptide is expressed in E. coli, e.g., strain PEB199.Expression of the GST—cytidine deaminase-like fusion protein in PEB199is induced with IPTG. The recombinant fusion polypeptide is purifiedfrom crude bacterial lysates of the induced PEB199 strain by affinitychromatography on glutathione beads. Using polyacrylamide gelelectrophoretic analysis of the polypeptide purified from the bacteriallysates, the molecular weight of the resultant fusion polypeptide isdetermined.

Example 3 Expression of Recombinant Cytidine Deaminase-Like Protein inCOS Cells

[0276] To express the cytidine deaminase-like gene in COS cells, thepcDNA/Amp vector by Invitrogen Corporation (San Diego, Calif.) is used.This vector contains an SV40 origin of replication, an ampicillinresistance gene, an E. coli replication origin, a CMV promoter followedby a polylinker region, and an SV40 intron and polyadenylation site. ADNA fragment encoding the entire cytidine deaminase-like protein and anHA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frameto its 3′ end of the fragment is cloned into the polylinker region ofthe vector, thereby placing the expression of the recombinant proteinunder the control of the CMV promoter.

[0277] To construct the plasmid, the cytidine deaminase-like DNAsequence is amplified by PCR using two primers. The 5′ primer containsthe restriction site of interest followed by approximately twentynucleotides of the cytidine deaminase-like coding sequence starting fromthe initiation codon; the 3′ end sequence contains complementarysequences to the other restriction site of interest, a translation stopcodon, the HA tag or FLAG tag and the last 20 nucleotides of thecytidine deaminase-like coding sequence. The PCR amplified fragment andthe pCDNA/Amp vector are digested with the appropriate restrictionenzymes and the vector is dephosphorylated using the CIAP enzyme (NewEngland Biolabs, Beverly, Mass.). Preferably the two restriction siteschosen are different so that the cytidine deaminase-like gene isinserted in the correct orientation. The ligation mixture is transformedinto E. coli cells (strains HB101, DH5α, SURE, available from StratageneCloning Systems, La Jolla, Calif., can be used), the transformed cultureis plated on ampicillin media plates, and resistant colonies areselected. Plasmid DNA is isolated from transformants and examined byrestriction analysis for the presence of the correct fragment.

[0278] COS cells are subsequently transfected with the cytidinedeaminase-like-pcDNA/Amp plasmid DNA using the calcium phosphate orcalcium chloride co-precipitation methods, DEAE-dextran-mediatedtransfection, lipofection, or electroporation. Other suitable methodsfor transfecting host cells can be found in Sambrook, J., Fritsh, E. F.,and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989. The expression of the cytidine deaminase-likepolypeptide is detected by radiolabelling (³⁵S-methionine or³⁵S-cysteine available from NEN, Boston, Mass., can be used) andimmunoprecipitation (Harlow, E. and Lane, D. Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1988) using an HA specific monoclonal antibody. Briefly, the cells arelabeled for 8 hours with ³⁵S-methionine (or ³⁵S-cysteine). The culturemedia are then collected and the cells are lysed using detergents (RIPAbuffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5).Both the cell lysate and the culture media are precipitated with an HAspecific monoclonal antibody. Precipitated polypeptides are thenanalyzed by SDS-PAGE.

[0279] Alternatively, DNA containing the cytidine deaminase-like codingsequence is cloned directly into the polylinker of the pCDNA/Amp vectorusing the appropriate restriction sites. The resulting plasmid istransfected into COS cells in the manner described above, and theexpression of the cytidine deaminase-like polypeptide is detected byradiolabelling and immunoprecipitation using a cytidine deaminase-likespecific monoclonal antibody.

[0280] All publications and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

[0281] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

1 4 1 1585 DNA Homo sapiens CDS (149)...(1165) misc_feature (1)...(1585)n = A,T,C or G 1 ngcggtcaag cttgaggcgt catctggctg cgcctagtgg gccgttgccttacagttgct 60 gagaggaggt gagaggcggg ggcgctaggg ccgagatcat gtctgactgggagaggtttc 120 cttggcagca gaggacgcta ggtttggg atg aaa gaa gct ggg cagatg caa 172 Met Lys Glu Ala Gly Gln Met Gln 1 5 aat ctg gag agc gcg agggcc ggg cgg tca gtc agc acc cag act ggc 220 Asn Leu Glu Ser Ala Arg AlaGly Arg Ser Val Ser Thr Gln Thr Gly 10 15 20 agc atg acc ggt cag ata ccaagg ctt tct aaa gtc aac ctt ttc act 268 Ser Met Thr Gly Gln Ile Pro ArgLeu Ser Lys Val Asn Leu Phe Thr 25 30 35 40 ctg ctc agc ctc tgg atg gagctc ttt cca gca gaa gcc cag cgg caa 316 Leu Leu Ser Leu Trp Met Glu LeuPhe Pro Ala Glu Ala Gln Arg Gln 45 50 55 aaa tct cag aaa aat gaa gag ggaaag cat gga ccc tta gga gat aat 364 Lys Ser Gln Lys Asn Glu Glu Gly LysHis Gly Pro Leu Gly Asp Asn 60 65 70 gaa gag agg acc aga gta tct act gacaaa aga cag gta aag aga act 412 Glu Glu Arg Thr Arg Val Ser Thr Asp LysArg Gln Val Lys Arg Thr 75 80 85 ggt ctt gtg gtg gtg aaa aac atg aaa attgtt ggt ctc cac tgt tct 460 Gly Leu Val Val Val Lys Asn Met Lys Ile ValGly Leu His Cys Ser 90 95 100 agt gaa gat tta cat gcc ggg cag att gctctt att aaa cat ggg tca 508 Ser Glu Asp Leu His Ala Gly Gln Ile Ala LeuIle Lys His Gly Ser 105 110 115 120 agg ctg aaa aac tgt gat ctt tat ttttcc aga aaa cca tgt tct gct 556 Arg Leu Lys Asn Cys Asp Leu Tyr Phe SerArg Lys Pro Cys Ser Ala 125 130 135 tgt ttg aaa atg att gta aat gct ggagtt aac cga att tca tac tgg 604 Cys Leu Lys Met Ile Val Asn Ala Gly ValAsn Arg Ile Ser Tyr Trp 140 145 150 cct gct gat cca gaa ata agt ttg cttacg gag gct tct agt tct gaa 652 Pro Ala Asp Pro Glu Ile Ser Leu Leu ThrGlu Ala Ser Ser Ser Glu 155 160 165 gat gca aag tta gat gcc aaa gca gtggaa aga ttg aag tca aac agt 700 Asp Ala Lys Leu Asp Ala Lys Ala Val GluArg Leu Lys Ser Asn Ser 170 175 180 cgg gcc cat gtg tgt gtc tta ctt caacct ttg gtg tgt tat atg gtg 748 Arg Ala His Val Cys Val Leu Leu Gln ProLeu Val Cys Tyr Met Val 185 190 195 200 cag ttt gta gag gag acc tct tacaaa tgt gac ttt att caa aaa att 796 Gln Phe Val Glu Glu Thr Ser Tyr LysCys Asp Phe Ile Gln Lys Ile 205 210 215 aca aaa aca ttg ccg gat gct aacact gac ttt tat tat gaa tgt aaa 844 Thr Lys Thr Leu Pro Asp Ala Asn ThrAsp Phe Tyr Tyr Glu Cys Lys 220 225 230 caa gaa aga ata aaa gaa tat gaaatg tta ttt ttg gtt tca aat gaa 892 Gln Glu Arg Ile Lys Glu Tyr Glu MetLeu Phe Leu Val Ser Asn Glu 235 240 245 gaa atg cat aag caa ata ctg atgact ata ggt ttg gag aac ctg tgt 940 Glu Met His Lys Gln Ile Leu Met ThrIle Gly Leu Glu Asn Leu Cys 250 255 260 gaa aat cca tac ttt agc aat ctaagg caa aac atg aaa gac ctt atc 988 Glu Asn Pro Tyr Phe Ser Asn Leu ArgGln Asn Met Lys Asp Leu Ile 265 270 275 280 cta ctt ttg gcc aca gta gcttcc agt gtg ccg aac ttt aaa cac ttc 1036 Leu Leu Leu Ala Thr Val Ala SerSer Val Pro Asn Phe Lys His Phe 285 290 295 gga ttt tac cgt agc aat ccagaa cag att aat gaa att cac aat caa 1084 Gly Phe Tyr Arg Ser Asn Pro GluGln Ile Asn Glu Ile His Asn Gln 300 305 310 agt ttg cca cag gaa att gcaagg cac tgc atg gtt cag gcc agg tta 1132 Ser Leu Pro Gln Glu Ile Ala ArgHis Cys Met Val Gln Ala Arg Leu 315 320 325 ttg gca tat cga act ggt gagtta cat aga tcg taaattgggg ctgattggtt 1185 Leu Ala Tyr Arg Thr Gly GluLeu His Arg Ser 330 335 gggttgtatt tgtctctgaa gtgttcgtct catttatggtagagttcatt tactcatagt 1245 tacttaagtt ttgctgttca tacaatatag agaagttagtgagacccttg agtagacaac 1305 tctttctccc agcagttttg ggattccttg tagccttatattcagtacca catttctaca 1365 tcaggccctc attaatctag gcccttcttt ctgcttcttgcttttatgat ttcactgktc 1425 cttgagccct ccactaaagg taggacaaga agagaaaggagaggcccagt gcagtggttc 1485 atgcctgtaa ttgcaacact ttagaaggct gadacaggaggatcgcttga gctcaggagt 1545 tcaagaccag cgtgggcaac atagcaagac ctcgactcta1585 2 339 PRT Homo sapiens 2 Met Lys Glu Ala Gly Gln Met Gln Asn LeuGlu Ser Ala Arg Ala Gly 1 5 10 15 Arg Ser Val Ser Thr Gln Thr Gly SerMet Thr Gly Gln Ile Pro Arg 20 25 30 Leu Ser Lys Val Asn Leu Phe Thr LeuLeu Ser Leu Trp Met Glu Leu 35 40 45 Phe Pro Ala Glu Ala Gln Arg Gln LysSer Gln Lys Asn Glu Glu Gly 50 55 60 Lys His Gly Pro Leu Gly Asp Asn GluGlu Arg Thr Arg Val Ser Thr 65 70 75 80 Asp Lys Arg Gln Val Lys Arg ThrGly Leu Val Val Val Lys Asn Met 85 90 95 Lys Ile Val Gly Leu His Cys SerSer Glu Asp Leu His Ala Gly Gln 100 105 110 Ile Ala Leu Ile Lys His GlySer Arg Leu Lys Asn Cys Asp Leu Tyr 115 120 125 Phe Ser Arg Lys Pro CysSer Ala Cys Leu Lys Met Ile Val Asn Ala 130 135 140 Gly Val Asn Arg IleSer Tyr Trp Pro Ala Asp Pro Glu Ile Ser Leu 145 150 155 160 Leu Thr GluAla Ser Ser Ser Glu Asp Ala Lys Leu Asp Ala Lys Ala 165 170 175 Val GluArg Leu Lys Ser Asn Ser Arg Ala His Val Cys Val Leu Leu 180 185 190 GlnPro Leu Val Cys Tyr Met Val Gln Phe Val Glu Glu Thr Ser Tyr 195 200 205Lys Cys Asp Phe Ile Gln Lys Ile Thr Lys Thr Leu Pro Asp Ala Asn 210 215220 Thr Asp Phe Tyr Tyr Glu Cys Lys Gln Glu Arg Ile Lys Glu Tyr Glu 225230 235 240 Met Leu Phe Leu Val Ser Asn Glu Glu Met His Lys Gln Ile LeuMet 245 250 255 Thr Ile Gly Leu Glu Asn Leu Cys Glu Asn Pro Tyr Phe SerAsn Leu 260 265 270 Arg Gln Asn Met Lys Asp Leu Ile Leu Leu Leu Ala ThrVal Ala Ser 275 280 285 Ser Val Pro Asn Phe Lys His Phe Gly Phe Tyr ArgSer Asn Pro Glu 290 295 300 Gln Ile Asn Glu Ile His Asn Gln Ser Leu ProGln Glu Ile Ala Arg 305 310 315 320 His Cys Met Val Gln Ala Arg Leu LeuAla Tyr Arg Thr Gly Glu Leu 325 330 335 His Arg Ser 3 1017 DNA Homosapiens 3 atgaaagaag ctgggcagat gcaaaatctg gagagcgcga gggccgggcggtcagtcagc 60 acccagactg gcagcatgac cggtcagata ccaaggcttt ctaaagtcaaccttttcact 120 ctgctcagcc tctggatgga gctctttcca gcagaagccc agcggcaaaaatctcagaaa 180 aatgaagagg gaaagcatgg acccttagga gataatgaag agaggaccagagtatctact 240 gacaaaagac aggtaaagag aactggtctt gtggtggtga aaaacatgaaaattgttggt 300 ctccactgtt ctagtgaaga tttacatgcc gggcagattg ctcttattaaacatgggtca 360 aggctgaaaa actgtgatct ttatttttcc agaaaaccat gttctgcttgtttgaaaatg 420 attgtaaatg ctggagttaa ccgaatttca tactggcctg ctgatccagaaataagtttg 480 cttacggagg cttctagttc tgaagatgca aagttagatg ccaaagcagtggaaagattg 540 aagtcaaaca gtcgggccca tgtgtgtgtc ttacttcaac ctttggtgtgttatatggtg 600 cagtttgtag aggagacctc ttacaaatgt gactttattc aaaaaattacaaaaacattg 660 ccggatgcta acactgactt ttattatgaa tgtaaacaag aaagaataaaagaatatgaa 720 atgttatttt tggtttcaaa tgaagaaatg cataagcaaa tactgatgactataggtttg 780 gagaacctgt gtgaaaatcc atactttagc aatctaaggc aaaacatgaaagaccttatc 840 ctacttttgg ccacagtagc ttccagtgtg ccgaacttta aacacttcggattttaccgt 900 agcaatccag aacagattaa tgaaattcac aatcaaagtt tgccacaggaaattgcaagg 960 cactgcatgg ttcaggccag gttattggca tatcgaactg gtgagttacatagatcg 1017 4 100 PRT Artificial Sequence PFAM consensus sequence forcytidine and deoxycytidylate deaminase zinc-binding region 4 Thr Pro TyrSer Gly Phe Pro Val Gly Ala Val Ile Val Lys Asp Asn 1 5 10 15 Gly ArgIle Phe Gly Val Asn Ser Glu Gly Ala Asn Tyr Val Glu Gly 20 25 30 Glu GlnLys Lys Asp Pro Thr Ala His Ala Glu Val Asn Ala Ile Arg 35 40 45 Lys AlaVal Ser Glu Arg Tyr Arg Asp Phe Lys Ile Arg Leu Gly Gly 50 55 60 Glu ArgLeu Glu Gly Ala Thr Leu Tyr Val Thr Leu Glu Pro Cys Gly 65 70 75 80 HisTyr Gly Arg Thr Pro Met Cys Ala Gln Ala Ile Ile Glu Ser Gly 85 90 95 IleLys Lys Val 100

That which is claimed:
 1. An isolated nucleic acid molecule selectedfrom the group consisting of: a) a nucleic acid molecule comprising anucleotide sequence which is at least 60% identical to the nucleotidesequence of SEQ ID NO:1 or SEQ ID NO:3, wherein said sequence encodes apolypeptide having biological activity; b) a nucleic acid moleculecomprising a fragment of at least 20 nucleotides of the nucleotidesequence of SEQ ID NO:1 or SEQ ID NO:3; c) a nucleic acid molecule whichencodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2;d) a nucleic acid molecule which encodes a fragment of a polypeptidecomprising the amino acid sequence of SEQ ID NO:2, wherein the fragmentcomprises at least 15 contiguous amino acids of SEQ ID NO:2; e) anucleic acid molecule which encodes a naturally occurring allelicvariant of a biologically active polypeptide comprising the amino acidsequence of SEQ ID NO:2, wherein the nucleic acid molecule hybridizes toa nucleic acid molecule comprising the complement of SEQ ID NO:1 or SEQID NO:3 under stringent conditions; and, f) a nucleic acid moleculecomprising the complement of a), b), c), d), or e).
 2. The isolatednucleic acid molecule of claim 1, which is selected from the groupconsisting of: a) a nucleic acid comprising the nucleotide sequence ofSEQ ID NO:1, SEQ ID NO:3, or complement thereof; and, b) a nucleic acidmolecule which encodes a polypeptide comprising the amino acid sequenceof SEQ ID NO:2.
 3. The nucleic acid molecule of claim 1 furthercomprising vector nucleic acid sequences.
 4. The nucleic acid moleculeof claim 1 further comprising nucleic acid sequences encoding aheterologous polypeptide.
 5. A host cell which contains the nucleic acidmolecule of claim
 1. 6. The host cell of claim 5 which is a mammalianhost cell.
 7. A non-human mammalian host cell containing the nucleicacid molecule of claim
 1. 8. An isolated polypeptide selected from thegroup consisting of: a) a biologically active polypeptide which isencoded by a nucleic acid molecule comprising a nucleotide sequencewhich is at least 60% identical to a nucleic acid comprising thenucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3; b) a naturallyoccurring allelic variant of a biologically active polypeptidecomprising the amino acid sequence of SEQ ID NO:2, wherein thepolypeptide is encoded by a nucleic acid molecule which hybridizes to anucleic acid molecule comprising the complement of SEQ ID NO:1 or SEQ IDNO:3 under stringent conditions; and, c) a fragment of a polypeptidecomprising the amino acid sequence of SEQ ID NO:2, wherein the fragmentcomprises at least 15 contiguous amino acids of SEQ ID NO:2; and, d) abiologically active polypeptide having at least 60% sequence identity tothe amino acid sequence SEQ ID NO:2.
 9. The isolated polypeptide ofclaim 8 comprising the amino acid sequence of SEQ ID NO:2.
 10. Thepolypeptide of claim 8 further comprising heterologous amino acidsequences.
 11. An antibody which selectively binds to a polypeptide ofclaim
 8. 12. A method for producing a polypeptide selected from thegroup consisting of: a) a polypeptide comprising the amino acid sequenceof SEQ ID NO:2; b) a polypeptide comprising a fragment of the amino acidsequence of SEQ ID NO:2, wherein the fragment comprises at least 15contiguous amino acids of SEQ ID NO:2; c) a naturally occurring allelicvariant of a biologically active polypeptide comprising the amino acidsequence of SEQ ID NO:2, wherein the polypeptide is encoded by a nucleicacid molecule which hybridizes to a nucleic acid molecule comprising thecomplement of SEQ ID NO:1 or 3; and, d) a biologically activepolypeptide having at least 60% sequence identity to the nucleic acidsequence of SEQ ID NO:2; comprising culturing a host cell underconditions in which the nucleic acid molecule is expressed.
 13. A methodfor detecting the presence of a polypeptide of claim 8 in a sample,comprising: a) contacting the sample with a compound which selectivelybinds to a polypeptide of claim 8; and b) determining whether thecompound binds to the polypeptide in the sample.
 14. The method of claim13, wherein said sample is derived from a subject having or predisposedto cancer.
 15. The method of claim 13, wherein the compound which bindsto the polypeptide is an antibody.
 16. A kit comprising a compound whichselectively binds to a polypeptide of claim 8 and instructions for use.17. A method for detecting the presence of a nucleic acid molecule ofclaim 1 in a sample, comprising the steps of: a) contacting the samplewith a nucleic acid probe or primer which selectively hybridizes to thenucleic acid molecule; and b) determining whether the nucleic acid probeor primer binds to a nucleic acid molecule in the sample.
 18. The methodof claim 17, wherein said sample is derived from a subject having orpredisposed to cancer.
 19. The method of claim 17, wherein the samplecomprises mRNA molecules and is contacted with a nucleic acid probe. 20.A kit comprising a compound which selectively hybridizes to a nucleicacid molecule of claim 1 and instructions for use.
 21. A method foridentifying a compound which binds to a polypeptide of claim 8comprising the steps of: a) contacting a polypeptide, or a cellexpressing a polypeptide of claim 8 with a test compound; and b)determining whether the polypeptide binds to the test compound.
 22. Themethod of claim 21, wherein the binding of the test compound to thepolypeptide is detected by a method selected from the group consistingof: a) detection of binding by direct detecting of testcompound/polypeptide binding; b) detection of binding using acompetition binding assay; c) detection of binding using an assay forcytidine deaminase-like mediated activity.
 23. A method for identifyinga compound which modulates the activity of a polypeptide of claim 8,comprising: a) contacting a polypeptide of claim 8 with a test compound;and b) determining the effect of the test compound on the activity ofthe polypeptide to thereby identify a compound that modulates theactivity of the polypeptide.
 24. A method for modulating the level oractivity of a nucleic acid molecule of claim 1, said method comprisingcontacting said nucleic acid molecule with an agent under conditionsthat allow the agent to modulate the level or activity of the nucleicacid molecule.
 25. The method of claim 24, wherein said host cell is ina subject having or predisposed to cancer.
 26. A method for modulatingthe level or activity of a polypeptide of claim 8 comprising contactinga polypeptide or a cell expressing a polypeptide of claim 8 with acompound which binds to the polypeptide in a sufficient concentration tomodulate the activity of the polypeptide.
 27. The method of claim 26,wherein said modulation is in a subject having or predisposed to cancer.28. A method for detecting a propensity of a subject to develop cancer,said method comprising obtaining a sample from said subject andcontacting said sample with an agent that specifically allows detectionof the presence of a nucleic acid molecule of claim 1 in the sample andthen detecting the presence of the nucleic acid molecule.